Methods and systems for sulfimidation or sulfoximidation of organic molecules

ABSTRACT

The disclosure generally relates to the fields of synthetic organic chemistry. In particular, the present disclosure relates to methods and systems for the imidation of sulfides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/625,514, filed Feb. 18, 2015 (now U.S. Pat. No. 9,399,762), whichclaims priority to U.S. Provisional Application No. 61/941,197, filedFeb. 18, 2014 and claims priority to U.S. Provisional Application No.61/976,927, filed Apr. 8, 2014, the disclosures of each of the foregoingapplication are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM101792 awardedby the National Institutes of Health and under N00014-11-1-0205 awardedby the Office of Naval Research. The government has certain rights inthe invention.

TECHNICAL FIELD

The present disclosure generally relates to the fields of syntheticorganic chemistry. In particular, the present disclosure relates tomethods and systems for the imidation of sulfides.

BACKGROUND

Enzymes offer appealing alternatives to traditional chemical catalystsdue to their ability to function in aqueous media at ambient temperatureand pressure. In addition, the ability of enzymes to orient substratebinding for defined regio- and stereo-chemical outcomes is highlyvaluable. This property is exemplified by the cytochrome P450monooxygenase family of enzymes that catalyze insertion of oxygen atomsinto unactivated C—H bonds (P. R. O. d. Montellano, Cytochrome P450:Structure, Mechanism and Biochemistry. Kluwer Academic/PlenumPublishers, New York, ed. 3rd Edition, 2005).

Cytochrome P450s catalyze monooxygenation with high degrees of regio-and stereo-selectivity, a property that makes them attractive for use inchemical synthesis. This broad enzyme class is capable of oxygenating awide variety of organic molecules including aromatic compounds, fattyacids, alkanes and alkenes. Diverse substrate selectivity is a hallmarkof this enzyme family and is exemplified in the natural world by theirimportance in natural product oxidation as well as xenobiotic metabolism(F. P. Guengerich, Chem. Res. Toxicol. 14, 611 (2001)). Limitations tothis enzyme class in synthesis include their large size, need forexpensive reducing equivalents (e.g., NADPH) and cellulardistribution—many cytochrome P450s are membrane bound and thereforedifficult to handle (Montellano, Cytochrome P450: Structure, Mechanismand Biochemistry. Kluwer Academic/Plenum Publishers, New York, ed. 3rdEdition, 2005). Several soluble bacterial cytochrome P450s have beenisolated, however, that show excellent properties and behavior forchemical synthesis and protein engineering applications.

SUMMARY

The disclosure provides method and compositions comprising one or moreheme enzymes that catalyze the nitrene transfer or insertion into anorganosulfur compounds comprising an —S— target site to form a new S—Nbond. In particular embodiments, the disclosure provides heme enzymevariants comprising at least one or more amino acid mutations thereinthat catalyze sulfoxidation and/or sulfimidation, making productsdescribed herein with high stereoselectivity. In some embodiments, theheme enzyme variants of the disclosure have the ability to catalyzenitrene transfer reactions efficiently, display increased total turnovernumbers, and/or demonstrate highly regio- and/or enantioselectiveproduct formation compared to the corresponding wild-type enzymes.

The disclosure provides a method for catalyzing the intermolecularinsertion of nitrogen into thioethers, sulfur-organo compounds orsulfoxides to produce a product having a new S—N bond, the methodcomprising providing a nitrene source, a thioether or sulfoxideprecursor and a heme enzyme or an engineered heme enzyme; and allowingthe reaction to proceed for a time sufficient to form a product having anew S—N bond. In one embodiment, the nitrene source is an azide. In afurther embodiment, the azide has the general formula R¹—N₃, wherein R¹is (i) a substituted or unsubstituted aryl, a substitute orunsubstituted alkyl, —OR², or —NR², wherein R² is a substituted orunsubstituted aryl, a substitute or unsubstituted alkyl; (ii) —SO₂R³,wherein R³ a substituted or unsubstituted aryl, a substitute orunsubstituted alkyl, —OR² or NR², wherein R² are any alkyl or aryl;(iii) —COR⁴ wherein R⁴ is a substituted or unsubstituted aryl, asubstitute or unsubstituted alkyl, —OR² or NR², wherein R² are any alkylor aryl; or (iv) —P(O)(OR⁵)(OR⁶), wherein R⁵ and R⁶ are independently H,a substituted or unsubstituted aryl, a substitute or unsubstitutedalkyl. In a further embodiment, the azide has a structure selected fromthe group consisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl. In another embodiment, the nitrene source is selectedfrom the group consisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl. In another embodiment, the S—N containing product is analiphatic amine and the nitrene precursor is tosyl azide. In yet anotherembodiment, the product is generated through a nitrenoid intermediate.In still yet another embodiment, the engineered heme enzyme is acytochrome P450 enzyme or a variant thereof. In a further embodiment,the cytochrome P450 enzyme is expressed in a bacterial, archaeal orfungal host organism. In yet another embodiment, the cytochrome P450enzyme is a P450 BM3 enzyme or a variant thereof. In a furtherembodiment, the cytochrome P450 BM3 enzyme comprises the amino acidsequence set forth in SEQ ID NO: 1 or a variant thereof. In yet otherembodiments of the foregoing the cytochrome P450 enzyme variantcomprises a mutation at the axial position of the heme coordinationsite. In a further embodiment, the mutation is an amino acidsubstitution of Cys with a member selected from the group consisting ofAla, Asp, Arg, Asn, Glu, Gln, Gly, His, He, Lys, Leu, Met, Phe, Pro,Ser, Thr, Trp, Tyr and Val at the axial position. In still a furtherembodiment, the mutation is an amino acid substitution of Cys with Aspor Ser at the axial position. In another embodiment of any of theforegoing, the P450 BM3 enzyme variant comprises at least one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve, or allthirteen of the following amino acid substitutions in SEQ ID NO: 1:V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,T268A, A290V,L353V, 1366V, and E442K. In another embodiment of any of the foregoingthe cytochrome P450 enzyme variant comprises a T268A mutation and/or aC400X mutation in SEQ ID NO: 1, wherein X is any amino acid other thanCys. In still another embodiment, the cytochrome P450 enzyme variantcomprises a T438S mutation and/or a C400X mutation in SEQ ID NO: 1,wherein X is any amino acid other than Cys. In yet another embodiment,the cytochrome P450 enzyme variant comprises a T268A mutation, a C400Xmutation and a T438S mutation in SEQ ID NO:1, wherein X is any aminoacid other than Cys. In another embodiment, the engineered heme enzymecomprises a fragment of the cytochrome P450 enzyme or variant thereof.In yet another embodiment, the engineered heme enzyme is a cytochromeP450 BM3 enzyme variant selected from Table 4, Table 5 and Table 6. Inyet another embodiment, the product is a compound of Formula 1a:

wherein R¹ is a sulfoxide, a carbonyl or a phosphonate; wherein R² is Hor any alkyl or aryl; and wherein R³ is H, O or an optionallysubstituted aryl group. In a further embodiment, R¹ is a sulfoxide offormula SO₂R⁵, wherein R⁵ any alkyl, any aryl, —OR⁶ or NR⁷, wherein R⁶and R⁷ are any alkyl or any aryl. In a further embodiment, R² is anyalkyl or aryl. In still a further embodiment, R³ is H. In yet anotherembodiment of Formula 1a R¹ is a phosphonate of formula P(O)(OR⁸)(OR⁹),wherein R⁸ and R⁹ are independently any aryl or any alkyl. In a furtherembodiment, R² is any alkyl or any aryl. In still a further embodiment,R³ is any alkyl or aryl. In yet another embodiment of Formula 1a, R¹ isa carbonyl group. In a further embodiment, R² is any alkyl or any aryl.In a further embodiment, R³ is any alkyl or any aryl. In anotherembodiment of Formula 1a, R³ is an optionally substituted aryl group. Ina further embodiment, R¹ is any alkyl or any aryl. In a furtherembodiment, R² is H or any alkyl or any aryl. In another embodiment ofFormula 1a, R¹ is a carbonyl group. In a further embodiment, R² is anyalkyl or any aryl. In a further embodiment, R³ is O.

The disclosure also provides products made by any of the foregoingmethods.

Also provided is a reaction mixture comprising a nitrene source, athioether or sulfoxide substrate and an engineered heme enzyme forproducing a product having a new S—N bond. In one embodiment, thenitrene source is an azide. In a further embodiment, the azide has thegeneral formula R¹—N₃, wherein R¹ is (i) a substituted or unsubstitutedaryl, a substitute or unsubstituted alkyl, —OR², or —NR², wherein R² isa substituted or unsubstituted aryl, a substitute or unsubstitutedalkyl; (ii) —SO₂R³, wherein R³ a substituted or unsubstituted aryl, asubstitute or unsubstituted alkyl, —OR² or NR², wherein R² are any alkylor aryl; (iii) —COR⁴ wherein R⁴ is a substituted or unsubstituted aryl,a substitute or unsubstituted alkyl, —OR² or NR², wherein R² are anyalkyl or aryl; or (iv) —P(O)(OR⁵)(OR⁶), wherein R⁵ and R⁶ areindependently H, a substituted or unsubstituted aryl, a substitute orunsubstituted alkyl. In a further embodiment, the azide has a structureselected from the group consisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl. In another embodiment, the nitrene source is selectedfrom the group consisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl. In another embodiment, the S—N containing product is analiphatic amine and the nitrene precursor is tosyl azide. In yet anotherembodiment, the product is generated through a nitrenoid intermediate.In still yet another embodiment, the engineered heme enzyme is acytochrome P450 enzyme or a variant thereof. In a further embodiment,the cytochrome P450 enzyme is expressed in a bacterial, archaeal orfungal host organism. In yet another embodiment, the cytochrome P450enzyme is a P450 BM3 enzyme or a variant thereof. In a furtherembodiment, the cytochrome P450 BM3 enzyme comprises the amino acidsequence set forth in SEQ ID NO: 1 or a variant thereof. In yet otherembodiments of the foregoing the cytochrome P450 enzyme variantcomprises a mutation at the axial position of the heme coordinationsite. In a further embodiment, the mutation is an amino acidsubstitution of Cys with a member selected from the group consisting ofAla, Asp, Arg, Asn, Glu, Gln, Gly, His, He, Lys, Leu, Met, Phe, Pro,Ser, Thr, Trp, Tyr and Val at the axial position. In still a furtherembodiment, the mutation is an amino acid substitution of Cys with Aspor Ser at the axial position. In another embodiment of any of theforegoing, the P450 BM3 enzyme variant comprises at least one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve, or allthirteen of the following amino acid substitutions in SEQ ID NO: 1:V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G,T268A, A290V,L353V, 1366V, and E442K. In another embodiment of any of the foregoingthe cytochrome P450 enzyme variant comprises a T268A mutation and/or aC400X mutation in SEQ ID NO: 1, wherein X is any amino acid other thanCys. In still another embodiment, the cytochrome P450 enzyme variantcomprises a T438S mutation and/or a C400X mutation in SEQ ID NO: 1,wherein X is any amino acid other than Cys. In yet another embodiment,the cytochrome P450 enzyme variant comprises a T268A mutation, a C400Xmutation and a T438S mutation in SEQ ID NO:1, wherein X is any aminoacid other than Cys. In another embodiment, the engineered heme enzymecomprises a fragment of the cytochrome P450 enzyme or variant thereof.In yet another embodiment, the engineered heme enzyme is a cytochromeP450 BM3 enzyme variant selected from Table 4, Table 5 and Table 6. Inyet another embodiment, the product is a compound of Formula 1a:

wherein R¹ is a sulfoxide, a carbonyl or a phosphonate; wherein R² is Hor any alkyl or aryl; and wherein R³ is H, O or an optionallysubstituted aryl group. In a further embodiment, R¹ is a sulfoxide offormula SO₂R⁵, wherein R⁵ any alkyl, any aryl, —OR⁶ or NR⁷, wherein R⁶and R⁷ are any alkyl or any aryl. In a further embodiment, R² is anyalkyl or aryl. In still a further embodiment, R³ is H. In yet anotherembodiment of Formula 1a R¹ is a phosphonate of formula P(O)(OR⁸)(OR⁹),wherein R⁸ and R⁹ are independently any aryl or any alkyl. In a furtherembodiment, R² is any alkyl or any aryl. In still a further embodiment,R³ is any alkyl or aryl. In yet another embodiment of Formula 1a, R¹ isa carbonyl group. In a further embodiment, R² is any alkyl or any aryl.In a further embodiment, R³ is any alkyl or any aryl. In anotherembodiment of Formula 1a, R³ is an optionally substituted aryl group. Ina further embodiment, R¹ is any alkyl or any aryl. In a furtherembodiment, R² is H or any alkyl or any aryl. In another embodiment ofFormula 1a, R¹ is a carbonyl group. In a further embodiment, R² is anyalkyl or any aryl. In a further embodiment, R³ is O.

The disclosure demonstrates the intermolecular nitrene transfercatalyzed by an enzyme, allowing for a mechanistic analysis of this newenzyme activity. Similar to P450-catalyzed sulfoxidation, the electronicproperties of the sulfide substrates influence reactivity, though themagnitude of the substituent effects is greater for nitrene transfer,possibly owing to the less oxidizing nature of the presumed nitrenoidintermediate.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description, serve toexplain the principles and implementations of the disclosure.

FIG. 1A-B shows P450-catalyzed sulfoxidation and P450-catalyzedsulfimination. (A) P450-catalyzed sulfoxidation, shown proceedingthrough compound I. This reaction can also be mediated by compound 0(hydroperoxy intermediate). (B) P411-catalyzed sulfimination proceedingthrough a nitrenoid intermediate formed from the azide with N₂ as abyproduct. Subsequent oxidation of the sulfilimine can result insulfoximine formation.

FIG. 2 shows a plot of reaction rate versus Hammett parameter ofsubstituted aryl sulfides in reactions using P411-CIS enzyme and tosylazide as nitrogen source. Data points are labeled with aryl substituentand position (p-=para, m-=meta).

FIG. 3 shows proposed mechanisms of sulfimide (“productive”) andsulfonamide (“unproductive”) formation.

FIG. 4 shows examples of azides tested as nitrene sources forsulfimidation of thioanisole using the P411_(BM3)-CIS T438S enzyme.

FIG. 5 shows examples of appropriate nitrene sources and generalizedreactions for the formation of sulfilimines.

FIG. 6 shows examples of appropriate nitrene sources and generalizedreactions for the formation of sulfoximines.

FIG. 7 shows data used to determine initial rates of the reactiondepicted in scheme above the graph for the enzymes listed in figurelegend.

FIG. 8A-D shows traces of (A) SFC trace of synthetic standard of 8ausing Chiralpak OD-H column with 25% isopropanol/75% supercritical CO₂mobile phase. (B) Trace of P411_(BM3)-CIS T438S produced 8a under sameconditions as synthetic standard. (C) Trace of P411_(BM3)-H2-5-F10produced 8a. (D) Trace of P411_(BM3)-CIS I263A T438S produced 8a, using20% isopropanol/80% supercritical CO₂ mobile phase.

FIG. 9 shows: Top: SFC trace of 8b synthetic standard using ChiralpakOD-H column with 20% isopropanol/80% supercritical CO₂ mobile phase.Bottom: Trace of enzyme produced 8b.

FIG. 10 shows: Top: SFC trace of 8c synthetic standard using ChiralpakOJ column with 15% isopropanol/85% supercritical CO₂ mobile phase.Bottom: Trace of enzyme produced 8c.

FIG. 11 shows: Top: SFC trace of 8d synthetic standard using ChiralpakOJ column with 15% isopropanol/85% supercritical CO₂ mobile phase.Bottom: Trace of enzyme produced 8d.

FIG. 12 shows Data used to determine initial rates for reaction ofsubstituted aryl sulfides with tosyl azide, using P411_(BM3)-CIS T438Sas catalyst. Sulfimide products measured are as listed in Table 2.

FIG. 13 shows visible spectroscopy of Fe(III) and Fe(II)-P411_(BM3)CISI263A T438S in the presence of NADPH, followed by azide addition.

FIG. 14 shows the monitoring the iron heme during the sulfimidationreaction with azide and sulfide. As shown in FIG. 13, the Soret peak forthe Fe(III) state is obscured by NADPH. Therefore the Q-band region(enlarged box from 530-640 nm) was used to assess the transition fromFe(III) to Fe(II) as reaction proceeds.

FIG. 15 shows the ratio of sulfimide TTN to sulfonamide TTN as sulfideconcentration is varied, using P411_(BM3)-CIS T438S enzyme, as shown inthe scheme. Reactions were prepared with azide concentration heldconstant at 1 mM and sulfide concentration varied from 0.5 mM to 4 mM.

FIG. 16 shows TTN values measured for slow addition vs. addingsubstrates simultaneously, using P411_(BM3)-CIS T438S enzyme and thesubstrates shown in FIG. S10. Light gray bar shows TTN for sulfonamide,9, dark gray shows TTN for the sulfimide, 8a.

FIG. 17 shows a standard curve for product 8a, shows in response factoras ratio of product area to internal standard area.

FIG. 18 shows the standard curve for product 8b.

FIG. 19 shows the standard curve for product 8c.

FIG. 20 shows the standard curve for product 8d.

FIG. 21 shows a demonstration of enzymatic production of 8a. Top: LC-MS230 nm chromatogram of synthetic standard of 8a, confirmed by NMR.Middle: Enzyme reaction containing putative 8a. Bottom: Mixture ofenzyme reaction and synthetic 8a, showing coelution.

FIG. 22 shows LC runs from FIG. 21 showing ESI-MS-(+) detection of totalion chromatogram (TIC) (major peak=324, corresponding to 8a M+H+).

FIG. 23 shows a demonstration of enzymatic production of 8b. Top: LC-MS230 nm chromatogram of synthetic standard of 8b, confirmed by NMR.Middle: Enzyme reaction containing putative 8b. Bottom: Mixture ofenzyme reaction and synthetic 8b, showing coelution.

FIG. 24 shows LC runs from FIG. 23 showing ESI-MS-(+) detection ofselected ions (mass window 307.5-308.5).

FIG. 25 shows a demonstration of enzymatic production of 8c. Top: LC-MS230 nm chromatogram of synthetic standard of 8c, confirmed by NMR.Middle: Enzyme reaction containing putative 8c. Bottom: Mixture ofenzyme reaction and synthetic 8c, showing coelution.

FIG. 26 shows LC runs from FIG. 25 showing ESI-MS-(+) detection ofselected ions (mass window 307.5-308.5).

FIG. 27 shows a demonstration of enzymatic production of 8d. Top: LC-MS230 nm chromatogram of synthetic standard of 8d, confirmed by NMR.Middle: Enzyme reaction containing putative 8d. Bottom: Mixture ofenzyme reaction and synthetic 8d, showing coelution.

FIG. 28 shows LC runs from FIG. 27 showing ESI-MS-(+) detection of totalion chromatogram (TIC) (major peak=294 m/z, corresponding to 8d M+H+).

FIG. 29 shows LC runs from reaction with P411_(BM3)-CIS T438S withsubstrate 7e showing ESIMS-(+) detection of selected ions (mass window321.5-322.5 m/z), demonstrating production of 8e.

FIG. 30 shows a mass spectrum of peak identified in FIG. 29, showingM+H+ of product 8e.

FIG. 31 shows an LC-MS chromatrogram showing UV trace at 230 nm (top)and selected ions for 344 m/z, corresponding to the M+H+ mass of theproduct of azide 2 with sulfide 7a. Note TIC timescale differs due toinstrument solvent delay.

FIG. 32 shows an LC-MS chromatrogram showing UV trace at 230 nm (top)and selected ions for 262 m/z, corresponding to the M+H+ mass of theproduct of azide 4 with sulfide 7a. Note TIC timescale differs due toinstrument solvent delay.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a species” includesa plurality of such species and reference to “the enzyme” includesreference to one or more enzymes and equivalents thereof, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure.

Enzymes offer many advantages over traditional catalysts, such asselectivity, mild reaction conditions, convenient production, and use inwhole cells. Cytochrome P450 enzymes are known to be able to carry outmonooxygenations of diverse substrates, and exemplify the mild operatingconditions that enzymes can afford. Many of the small molecule catalystsdeveloped for C—H amination reaction have been designed in an effort tomimic these enzymes, but with the goal of activating nitrene equivalentsrather than the oxene equivalents activated by cytochrome P450 enzymes(Bennett, R. D. & Heftmann, Phytochemistry 4, 873-879 (1965)).Cytochrome P450 enzymes bind to a cofactor comprising a catalytictransition metal (iron heme) that forms a reactive intermediate similarin electronic and steric features to metallonitrenoid intermediates usedfor synthetic C—N bond forming reactions.

The disclosure is based on the surprising discovery that engineered hemeenzymes such as cytochrome P450BM3 enzymes, including aserine-heme-ligated P411 enzyme, efficiently catalyze nitrene insertionand transfer reactions. Suitable reactions include, but are not limitedto, transfer of a nitrogen atom derived from an appropriate nitreneprecursor to sulfur atoms with formation of an S—N bond. For example, incertain aspects, the present disclosure provides engineered heme enzymessuch as cytochrome P450BM3 enzymes, including the serine-heme-ligated‘P411’, which efficiently catalyze the sulfimidation of variousorgansulfur molecules. Significant enhancements in catalytic activityand enantioselectivity are observed in vivo, using intact bacterialcells expressing the engineered enzymes. The results presented hereunderscore the utility of enzymes in catalyzing new reaction types withthe aid of synthetic reagents. The ability to genetically encodecatalysts for formal nitrene transfers opens up new biosyntheticpathways to amines and expands the scope of transformations accessibleto biocatalysis.

The term “S—N sulfimidation” includes a transfer of a nitrogen atomderived from an appropriate nitrene precursor to sulfur atoms withformation of an S—N bond, yielding a sulfimide.

The term “S—N sulfimidation (enzyme) catalyst” or “enzyme with S—Nsuflimidation activity” includes any and all chemical processescatalyzed by enzymes, by which substrates containing at least onecarbon-sulfur bond can be converted into sulfimide products by usingnitrene precursors such as sulfonyl azides, carbonyl azides, arylazides, azidoformates, phosphoryl azides, azide phosphonates,iminoiodanes, or haloamine derivatives.

This disclosure describes enzyme catalysts based for the transfer ofnitrogen atoms to aryl sulfides and other organosulfur compounds. Thisreaction is presumed to take place through a metal-nitrenoidintermediate, the reactivity of which is modulated by both the enzymeand substrates.

In some embodiments the organosulfur molecule has the structure offormula (I):

in which X═S atom is a target site for addition of a nitrogen, and R₁,R₂, and R₃ are independently selected from the group consisting ofhydrogen, oxygen, aliphatic, aryl, substituted aliphatic, substitutedaryl, heteroatom-containing aliphatic, heteroatom-containing aryl,substituted heteroatom-containing aliphatic, substitutedheteroatom-containing aryl, alkoxy, aryloxy, and functional groups (FG)or are taken together to form a ring.

The term “aliphatic” is used in the conventional sense to refer to anopen-chain or cyclic, linear or branched, saturated or unsaturatedhydrocarbon group, including but not limited to alkyl group, alkenylgroup and alkynyl groups. The term “heteroatom-containing aliphatic” asused herein refer to an aliphatic moiety where at least one carbon atomis replaced with a heteroatom.

The term “alkyl” and “alkyl group” as used herein refers to a linear,branched, or cyclic saturated hydrocarbon typically containing 1 to 24carbon atoms, preferably 1 to 12 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl and thelike. The term “heteroatom-containing alkyl” as used herein refers to analkyl moiety where at least one carbon atom is replaced with aheteroatom, e.g. oxygen, nitrogen, sulphur, phosphorus, or silicon, andtypically oxygen, nitrogen, or sulphur.

The term “alkenyl” and “alkenyl group” as used herein refers to alinear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms,preferably of 2 to 12 carbon atoms, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, and the like. The term “heteroatom-containing alkenyl”as used herein refer to an alkenyl moiety where at least one carbon atomis replaced with a heteroatom.

The term “alkynyl” and “alkynyl group” as used herein refers to alinear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms,preferably of 2 to 12 carbon atoms, containing at least one triple bond,such as ethynyl, n-propynyl, and the like. The term“heteroatom-containing alkynyl” as used herein refer to an alkynylmoiety where at least one carbon atom is replaced with a heteroatom.

The term “aryl” and “aryl group” as used herein refers to an aromaticsubstituent containing a single aromatic or multiple aromatic rings thatare fused together, directly linked, or indirectly linked (such aslinked through a methylene or an ethylene moiety). Preferred aryl groupscontain 5 to 24 carbon atoms, and particularly preferred aryl groupscontain 5 to 14 carbon atoms. The term “heteroatom-containing aryl” asused herein refer to an aryl moiety where at least one carbon atom isreplaced with a heteroatom.

The term “alkoxy” and “alkoxy group” as used herein refers to analiphatic group or a heteroatom-containing aliphatic group bound througha single, terminal ether linkage. Preferred aryl alkoxy groups contain 1to 24 carbon atoms, and particularly preferred alkoxy groups contain 1to 14 carbon atoms.

The term “aryloxy” and “aryloxy group” as used herein refers to an arylgroup or a heteroatom-containing aryl group bound through a single,terminal ether linkage. Preferred aryloxy groups contain 5 to 24 carbonatoms, and particularly preferred aryloxy groups contain 5 to 14 carbonatoms.

The terms “halo” and “halogen” are used in the conventional sense torefer to a fluoro, chloro, bromo or iodo substituent.

By “substituted” it is intended that in the alkyl, alkenyl, alkynyl,aryl, or other moiety, at least one hydrogen atom is replaced with oneor more non-hydrogen atoms. Examples of such substituents include,without limitation: functional groups referred to herein as “FG”, suchas alkoxy, aryloxy, alkyl, heteroatom-containing alkyl, alkenyl,heteroatom-containing alkenyl, alkynyl, heteroatom-containing alkynyl,aryl, heteroatom-containing aryl, alkoxy, heteroatom-containing alkoxy,aryloxy, heteroatom-containing aryloxy, halo, hydroxyl (—OH), sulfhydryl(—SH), substituted sulfhydryl, carbonyl (—CO—), thiocarbonyl, (—CS—),carboxy (—COOH), amino (—NH₂), substituted amino, nitro (—NO₂), nitroso(—NO), sulfo (—SO₂—OH), cyano (—C≡N), cyanato (—O—C≡N), thiocyanato(—S—C≡N), formyl (—CO—H), thioformyl (—CS—H), phosphono (—P(O)OH₂),substituted phosphono, and phospho (—PO₂).

In particular, the substituents R₁, R₂ and R₃ of formula I can beindependently selected from hydrogen, C₁-C₂₄ alkyl, C₁-C₂₄ substitutedalkyl, C₁-C₂₄ substituted heteroatom-containing alkyl, C₁-C₂₄substituted heteroatom-containing alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄substituted alkenyl, C₂-C₂₄ substituted heteroatom-containing alkenyl,C₂-C₂₄ substituted heteroatom-containing alkenyl, C₅-C₂₄ aryl, C₅-C₂₄substituted aryl, C₅-C₂₄ substituted heteroatom-containing aryl, C₅-C₂₄substituted heteroatom-containing aryl, C₁-C₂₄ alkoxy, C₅-C₂₄ aryloxy,carbonyl, thiocarbonyl, and carboxy. More in particular, R₁, R₂ and R₃of formula I can be independently selected from hydrogen, C₁-C₁₂ alkyl,C₁-C₁₂ substituted alkyl, C₁-C₁₂ substituted heteroatom-containingalkyl, C₁-C₁₂ substituted heteroatom-containing alkyl, C₂-C₁₂ alkenyl,C₂-C₁₂ substituted alkenyl, C₂-C₁₂ substituted heteroatom-containingalkenyl, C₂-C₁₂ substituted heteroatom-containing alkenyl, C₅-C₁₄ aryl,C₅-C₁₄ substituted aryl, C₅-C₁₄ substituted heteroatom-containing aryl,C₅-C₁₄ substituted heteroatom-containing aryl, C₂-C₁₄ alkoxy, C₅-C₁₄aryloxy, carbonyl, thiocarbonyl, and carboxy.

As used herein, the term “alkylthio” refers to an alkyl group having asulfur atom that connects the alkyl group to the point of attachment:i.e., alkyl-S—. As for alkyl groups, alkylthio groups can have anysuitable number of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkylthio groupsinclude, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.Alkylthio groups can be optionally substituted with one or more moietiesselected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, and cyano. Thioethers have the generalsstructure R—S—R, wherein R is any alkyl, alkynyl, alkenyl, aryl(substituted or unsubstituted).

As used herein, the term “haloalkyl” refers to an alkyl moiety asdefined above substituted with at least one halogen atom.

As used herein, the term “acyl” refers to a moiety —C(O)R, wherein R isan alkyl group.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

As used herein, the term “carboxy” refers to a moiety —C(O)OH. Thecarboxy moiety can be ionized to form the carboxylate anion.

As used herein, the term “amino” refers to a moiety —NR³, wherein each Rgroup is H or alkyl.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR², wherein each R group is H or alkyl.

The terms “engineered heme enzyme” and “heme enzyme variant” include anyheme-containing enzyme comprising at least one amino acid mutation withrespect to wild-type and also include any chimeric protein comprisingrecombined sequences or blocks of amino acids from two, three, or moredifferent heme-containing enzymes that will improve its S—Nsulfimidation activity or other reactions disclosed herein.

The terms “engineered cytochrome P450” and “cytochrome P450 variant”include any cytochrome P450 enzyme comprising at least one amino acidmutation with respect to wild-type and also include any chimeric proteincomprising recombined sequences or blocks of amino acids from two,three, or more different cytochrome P450 enzymes.

As used herein, the term “whole cell catalyst” includes microbial cellsexpressing heme containing enzymes, engineered cytochrome P450, or acytochrome P450 variant, where the whole cell displays sulfimidationactivity or sulfoximdation activity.

As used herein, the term “nitrene equivalent” or “nitrene precursor”includes molecules that can be decomposed in the presence of metal (orenzyme) catalysts to structures that contain at least one monovalentnitrogen atom with only 6 valence shell electrons and that can betransferred to a sulfur to form sulfilimines or sulfoximines.

As used herein, the terms “microbial,” “microbial organism” and“microorganism” include any organism that exists as a microscopic cellthat is included within the domains of archaea, bacteria or eukarya.Therefore, the term is intended to encompass prokaryotic or eukaryoticcells or organisms having a microscopic size and includes bacteria,archaea and eubacteria of all species as well as eukaryoticmicroorganisms such as yeast and fungi. Also included are cell culturesof any species that can be cultured for the production of a chemical.

As used herein, the term “non-naturally occurring,” when used inreference to a microbial organism or enzyme activity of the disclosure,is intended to mean that the microbial organism or enzyme has at leastone genetic alteration not normally found in a naturally occurringstrain of the referenced species, including wild-type strains of thereferenced species. Genetic alterations include, for example,modifications introducing expressible nucleic acids encoding metabolicpolypeptides, other nucleic acid additions, nucleic acid deletionsand/or other functional disruption of the microbial organism's geneticmaterial. Such modifications include, for example, coding regions andfunctional fragments thereof, for heterologous, homologous or bothheterologous and homologous polypeptides for the referenced species.Additional modifications include, for example, non-coding regulatoryregions in which the modifications alter expression of a gene or operon.

As used herein, the term “anaerobic”, when used in reference to areaction, culture or growth condition, is intended to mean that theconcentration of oxygen is less than about 25 μM, typically less thanabout 5 μM, and commonly less than 1 μM. The term is also intended toinclude sealed chambers of liquid or solid medium maintained with anatmosphere of less than about 1% oxygen. Typically, anaerobic conditionsare achieved by sparging a reaction mixture with an inert gas such asnitrogen or argon.

As used herein, the term “exogenous” is intended to mean that thereferenced molecule or the referenced activity is introduced into thehost microbial organism. The term as it is used in reference toexpression of an encoding nucleic acid refers to the introduction of theencoding nucleic acid in an expressible form into the microbial organismusing recombinant DNA techniques. When used in reference to abiosynthetic activity, the term refers to an activity that is introducedinto the host reference organism.

The term “heterologous” as used herein with reference to molecules, andin particular enzymes and polynucleotides, indicates molecules that areexpressed in an organism other than the organism from which theyoriginated or are found in nature, independently of the level ofexpression that can be lower, equal or higher than the level ofexpression of the molecule in the native microorganism.

On the other hand, the term “native” or “endogenous” as used herein withreference to molecules, and in particular enzymes and polynucleotides,indicates molecules that are expressed in the organism in which theyoriginated or are found in nature, independently of the level ofexpression that can be lower equal or higher than the level ofexpression of the molecule in the native microorganism. It is understoodthat expression of native enzymes or polynucleotides may be modified inrecombinant microorganisms.

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural and/or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In particular embodiments,the homology between two proteins is indicative of its shared ancestry,related by evolution.

The terms “analog” and “analogous” include nucleic acid or proteinsequences or protein structures that are related to one another infunction only and are not from common descent or do not share a commonancestral sequence. Analogs may differ in sequence but may share asimilar structure, due to convergent evolution. For example, two enzymesare analogs or analogous if the enzymes catalyze the same reaction ofconversion of a substrate to a product, are unrelated in sequence, andirrespective of whether the two enzymes are related in structure.

The term “contact” as used herein with reference to interactions ofchemical units indicates that the chemical units are at a distance thatallows short range non-covalent interactions (such as Van der Waalsforces, hydrogen bonding, hydrophobic interactions, electrostaticinteractions, dipole-dipole interactions) to dominate the interaction ofthe chemical units. For example, when an oxygenase enzyme is ‘contacted’with a target molecule, the enzyme is allowed to interact with and bindto the organic molecule through non-covalent interactions so that areaction between the enzyme and the target molecule can occur.

The term “introducing” as used herein with reference to the interactionbetween two chemical units, such as a functional groups and a targetsite, indicates a reaction resulting in the formation of a bond betweenthe two chemical units, e.g. the functional group and the target site.

The disclosure provides an enzymatic process for the suflimidation orsulfoximidation of a atarget sulfur atom b a nitrene precursor. Themethods and compositions of the disclosure includes a heme enzyme (e.g.,an engineered P450BM3 of variant) an nitrene precursor (e.g., an azideor other compound that comprises a nitrogen atom that can be used as anitrene) and a target organosulfur compound, wherein the compoundcomprises a sulfur atom that is linked to a nitrogen to form a new N-Cbond using the enzymatic process of the disclosure.

In some embodiments, the enzyme is a heme-containing enzyme or a variantthereof. The wording “heme” or “heme domain” as used herein refers to anamino acid sequence within an enzyme, which is capable of binding aniron-complexing structure such as a porphyrin. Compounds of iron aretypically complexed in a porphyrin (tetrapyrrole) ring that may differin side chain composition. Heme groups can be the prosthetic groups ofcytochromes and are found in most oxygen carrier proteins. Exemplaryheme domains include that of P450_(BM3) as well as truncated or mutatedversions of these that retain the capability to bind the iron-complexingstructure. A skilled person can identify the heme domain of a specificprotein using methods known in the art.

The terms “heme enzyme” and “heme protein” are used herein to includeany member of a group of proteins containing heme as a prosthetic group.Non-limiting examples of heme enzymes include globins, cytochromes,oxidoreductases, any other protein containing a heme as a prostheticgroup, and combinations thereof. Heme-containing globins include, butare not limited to, hemoglobin, myoglobin, and combinations thereof.Heme-containing cytochromes include, but are not limited to, cytochromeP450, cytochrome b, cytochrome cl, cytochrome c, and combinationsthereof. Heme-containing oxidoreductases include, but are not limitedto, a catalase, an oxidase, an oxygenase, a haloperoxidase, aperoxidase, and combinations thereof.

In certain instances, the heme enzymes are metal-substituted hemeenzymes containing protoporphyrin IX or other porphyrin moleculescontaining metals other than iron, including, but not limited to,cobalt, rhodium, copper, ruthenium, and manganese, which are activesulfimidation or sulfoximidation catalysts.

In some embodiments, the heme enzyme is a member of one of the enzymeclasses set forth in Table 1. In other embodiments, the heme enzyme is avariant or homolog of a member of one of the enzyme classes set forth inTable 1. In yet other embodiments, the heme enzyme comprises or consistsof the heme domain of a member of one of the enzyme classes set forth inTable 1 or a fragment thereof (e.g., a truncated heme domain) that iscapable of carrying out the nitrene insertion and nitrene transferreactions described herein.

TABLE 1 Heme enzymes identified by their enzyme classification number(EC number) and classification name. EC Number Name 1.1.2.3 L-lactatedehydrogenase 1.1.2.6 polyvinyl alcohol dehydrogenase (cytochrome)1.1.2.7 methanol dehydrogenase (cytochrome c) 1.1.5.5 alcoholdehydrogenase (quinone) 1.1.5.6 formate dehydrogenase-N: 1.1.9.1 alcoholdehydrogenase (azurin): 1.1.99.3 gluconate 2-dehydrogenase (acceptor)1.1.99.11 fructose 5-dehydrogenase 1.1.99.18 cellobiose dehydrogenase(acceptor) 1.1.99.20 alkan-1-ol dehydrogenase (acceptor) 1.2.1.70glutamyl-tRNA reductase 1.2.3.7 indole-3-acetaldehyde oxidase 1.2.99.3aldehyde dehydrogenase (pyrroloquinoline-quinone) 1.3.1.6 fumaratereductase (NADH): 1.3.5.1 succinate dehydrogenase (ubiquinone) 1.3.5.4fumarate reductase (menaquinone) 1.3.99.1 succinate dehydrogenase1.4.9.1 methylamine dehydrogenase (amicyanin) 1.4.9.2. aralkylaminedehydrogenase (azurin) 1.5.1.20 methylenetetrahydrofolate reductase[NAD(P)H] 1.5.99.6 spermidine dehydrogenase 1.6.3.1 NAD(P)H oxidase1.7.1.1 nitrate reductase (NADH) 1.7.1.2 Nitrate reductase [NAD(P)H]1.7.1.3 nitrate reductase (NADPH) 1.7.1.4 nitrite reductase [NAD(P)H]1.7.1.14 nitric oxide reductase [NAD(P), nitrous oxide-forming] 1.7.2.1nitrite reductase (NO-forming) 1.7.2.2 nitrite reductase (cytochrome;ammonia-forming) 1.7.2.3 trimethylamine-N-oxide reductase (cytochrome c)1.7.2.5 nitric oxide reductase (cytochrome c) 1.7.2.6 hydroxylaminedehydrogenase 1.7.3.6 hydroxylamine oxidase (cytochrome) 1.7.5.1 nitratereductase (quinone) 1.7.5.2 nitric oxide reductase (menaquinol) 1.7.6.1nitrite dismutase 1.7.7.1 ferredoxin-nitrite reductase 1.7.7.2ferredoxin-nitrate reductase 1.7.99.4 nitrate reductase 1.7.99.8hydrazine oxidoreductase 1.8.1.2 sulfite reductase (NADPH) 1.8.2.1sulfite dehydrogenase 1.8.2.2 thiosulfate dehydrogenase 1.8.2.3sulfide-cytochrome-c reductase (flavocytochrome c) 1.8.2.4 dimethylsulfide:cytochrome c2 reductase 1.8.3.1 sulfite oxidase 1.8.7.1 sulfitereductase (ferredoxin) 1.8.98.1 CoB-CoM heterodisulfide reductase1.8.99.1 sulfite reductase 1.8.99.2 adenylyl-sulfate reductase 1.8.99.3hydrogensulfite reductase 1.9.3.1 cytochrome-c oxidase 1.9.6.1 nitratereductase (cytochrome) 1.10.2.2 ubiquinol-cytochrome-c reductase1.10.3.1 catechol oxidase 1.10.3.B1 caldariellaquinol oxidase(H+-transporting) 1.10.3.3 L-ascorbate oxidase 1.10.3.9 photosystem II1.10.3.10 ubiquinol oxidase (H+-transporting) 1.10.3.11 ubiquinoloxidase 1.10.3.12 menaquinol oxidase (H+-transporting) 1.10.9.1plastoquinol-plastocyanin reductase 1.11.1.5 cytochrome-c peroxidase1.11.1.6 catalase 1.11.1.7 peroxidase 1.11.1.B2 chloride peroxidase(vanadium-containing) 1.11.1.B7 bromide peroxidase (heme-containing)1.11.1.8 iodide peroxidase 1.11.1.10 chloride peroxidase 1.11.1.11L-ascorbate peroxidase 1.11.1.13 manganese peroxidase 1.11.1.14 ligninperoxidase 1.11.1.16 versatile peroxidase 1.11.1.19 dye decolorizingperoxidase 1.11.1.21 catalase-peroxidase 1.11.2.1 unspecificperoxygenase 1.11.2.2 myeloperoxidase 1.11.2.3 plant seed peroxygenase1.11.2.4 fatty-acid peroxygenase 1.12.2.1 cytochrome-c3 hydrogenase1.12.5.1 hydrogen:quinone oxidoreductase 1.12.99.6 hydrogenase(acceptor) 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase 1.13.11.11tryptophan 2,3-dioxygenase 1.13.11.49 chlorite O2-lyase 1.13.11.50acetylacetone-cleaving enzyme 1.13.11.52 indoleamine 2,3-dioxygenase1.13.11.60 linoleate 8R-lipoxygenase 1.13.99.3 tryptophan 2′-dioxygenase1.14.11.9 flavanone 3-dioxygenase 1.14.12.17 nitric oxide dioxygenase1.14.13.39 nitric-oxide synthase (NADPH dependent) 1.14.13.17cholesterol 7alpha-monooxygenase 1.14.13.41 tyrosine N-monooxygenase1.14.13.70 sterol 14alpha-demethylase 1.14.13.71 N-methylcoclaurine3′-monooxygenase 1.14.13.81 magnesium-protoporphyrin IX monomethyl ester(oxidative) cyclase 1.14.13.86 2-hydroxyisoflavanone synthase 1.14.13.98cholesterol 24-hydroxylase 1.14.13.119 5-epiaristolochene1,3-dihydroxylase 1.14.13.126 vitamin D3 24-hydroxylase 1.14.13.129beta-carotene 3-hydroxylase 1.14.13.141 cholest-4-en-3-one26-monooxygenase 1.14.13.142 3-ketosteroid 9alpha-monooxygenase1.14.13.151 linalool 8-monooxygenase 1.14.13.156 1,8-cineole2-endo-monooxygenase 1.14.13.159 vitamin D 25-hydroxylase 1.14.14.1unspecific monooxygenase 1.14.15.1 camphor 5-monooxygenase 1.14.15.6cholesterol monooxygenase (side-chain-cleaving) 1.14.15.8 steroid15beta-monooxygenase 1.14.15.9 spheroidene monooxygenase 1.14.18.1tyrosinase 1.14.19.1 stearoyl-CoA 9-desaturase 1.14.19.3 linoleoyl-CoAdesaturase 1.14.21.7 biflaviolin synthase 1.14.99.1prostaglandin-endoperoxide synthase 1.14.99.3 heme oxygenase 1.14.99.9steroid 17alpha-monooxygenase 1.14.99.10 steroid 21-monooxygenase1.14.99.15 4-methoxybenzoate monooxygenase (O-demethylating) 1.14.99.45carotene epsilon-monooxygenase 1.16.5.1 ascorbate ferrireductase(transmembrane) 1.16.9.1 iron:rusticyanin reductase 1.17.1.4 xanthinedehydrogenase 1.17.2.2 lupanine 17-hydroxylase (cytochrome c) 1.17.99.14-methylphenol dehydrogenase (hydroxylating) 1.17.99.2 ethylbenzenehydroxylase 1.97.1.1 chlorate reductase 1.97.1.9 selenate reductase2.7.7.65 diguanylate cyclase 2.7.13.3 histidine kinase 3.1.4.52cyclic-guanylate-specific phosphodiesterase 4.2.1.B9 colneleicacid/etheroleic acid synthase 4.2.1.22 Cystathionine beta-synthase4.2.1.92 hydroperoxide dehydratase 4.2.1.212 colneleate synthase4.3.1.26 chromopyrrolate synthase 4.6.1.2 guanylate cyclase 4.99.1.3sirohydrochlorin cobaltochelatase 4.99.1.5 aliphatic aldoximedehydratase 4.99.1.7 phenylacetaldoxime dehydratase 5.3.99.3prostaglandin-E synthase 5.3.99.4 prostaglandin-I synthase 5.3.99.5Thromboxane-A synthase 5.4.4.5 9,12-octadecadienoate 8-hydroperoxide8R-isomerase 5.4.4.6 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase6.6.1.2 cobaltochelatase

In some embodiments, the heme enzyme is a variant or a fragment thereof(e.g., a truncated variant containing the heme domain) comprising atleast one mutation such as, e.g., a mutation at the axial position ofthe heme coordination site. In some instances, the mutation is asubstitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu,Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Valat the axial position. In certain instances, the mutation is asubstitution of Cys with any other amino acid such as Ser at the axialposition.

In certain embodiments, the in vitro methods for producing a productdescribed herein comprise providing a heme enzyme, variant, or homologthereof with a reducing agent such as NADPH or a dithionite salt (e.g.,Na₂S₂O₄). In certain other embodiments, the in vivo methods forproducing a reaction product provided herein comprise providing wholecells such as E. coli cells expressing a heme enzyme, variant, orhomolog thereof.

In some embodiments, the heme enzyme, variant, or homolog thereof isrecombinantly expressed and optionally isolated and/or purified forcarrying out the in vitro sulfimidation or sulfoximidation reactions ofthe disclosure. In other embodiments, the heme enzyme, variant, orhomolog thereof is expressed in whole cells such as E. coli cells, andthese cells are used for carrying out the in vivo nitrene insertionactivity and/or nitrene transfer activity of the disclosure.

In certain embodiments, the heme enzyme, variant, or homolog thereofcomprises or consists of the same number of amino acid residues as thewild-type enzyme (i.e., a full-length polypeptide). In some instances,the heme enzyme, variant, or homolog thereof comprises or consists of anamino acid sequence without the start methionine (e.g., P450BM3 aminoacid sequence set forth in SEQ ID NO:1). In other embodiments, the hemeenzyme comprises or consists of a heme domain fused to a reductasedomain. In yet other embodiments, the heme enzyme does not contain areductase domain, e.g., the heme enzyme contains a heme domain only or afragment thereof such as a truncated heme domain.

In some embodiments, the heme enzyme, variant, or homolog thereof has anenhanced nitrene insertion activity and/or nitrene transfer activity ofabout 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold compared to thecorresponding wild-type heme enzyme.

In some embodiments, the heme enzyme comprises a heme domain fused to areductase domain. In other embodiments, the heme enzyme does notcomprise a reductase domain, e.g., a heme domain only or a fragmentthereof.

In one embodiment, the disclosure provides a method for catalyzing anitrene insertion into a —S— bond to produce a product having a new S—Nbond. The method comprises the steps of: providing a —S— containingsubstrate, a nitene precursor and an engineered heme enzyme; andallowing the reaction to proceed for a time sufficient to form a producthaving a new S—N bond.

In particular embodiments, the heme enzyme comprises a cyctochrome P450enzyme. Cytochrome P450 enzymes constitute a large superfamily ofheme-thiolate proteins involved in the metabolism of a wide variety ofboth exogenous and endogenous compounds. Usually, they act as theterminal oxidase in multicomponent electron transfer chains, such asP450-containing monooxygenase systems. Members of the cytochrome P450enzyme family catalyze myriad oxidative transformations, including,e.g., hydroxylation, epoxidation, oxidative ring coupling, heteratomrelease, and heteroatom oxygenation (E. M. Isin et al., Biochim.Biophys. Acta 1770, 314 (2007)). The active site of these enzymescontains a Fe^(III)-protoporphyrin IX cofactor (heme) ligated proximallyby a conserved cysteine thiolate (M. T. Green, Current Opinion inChemical Biology 13, 84 (2009)). The remaining axial iron coordinationsite is occupied by a water molecule in the resting enzyme, but duringnative catalysis, this site is capable of binding molecular oxygen. Inthe presence of an electron source, typically provided by NADH or NADPHfrom an adjacent fused reductase domain or an accessory cytochrome P450reductase enzyme, the heme center of cytochrome P450 activates molecularoxygen, generating a high valent iron(IV)-oxo porphyrin cation radicalspecies intermediate and a molecule of water.

In some embodiments, the engineered heme enzyme is a cytochrome P450enzyme or a variant thereof. In certain embodiments, the P450 enzyme isa member of one of the classes shown in Table 2 (see, e.g.,http[://www].icegeb.org/˜p450srv/P450enzymes.html), the disclosure ofwhich is incoproated herein by reference in its entirety.

TABLE 2 Heme enzymes identified by their enzyme classification number(EC number) and classification name. EC Recommended name Family/gene1.3.3.9 secologanin synthase CYP72A1 1.14.13.11 trans-cinnamate4-monooxygenase CYP73 1.14.13.12 benzoate 4-monooxygenase CYP531.14.13.13 calcidiol 1-monooxygenase CYP27 1.14.13.15 cholestanetriol26-monooxygenase CYP27 1.14.13.17 cholesterol 7α-monooxygenase CYP71.14.13.21 flavonoid 3′-monooxygenase CYP75 1.14.13.283,9-dihydroxypterocarpan 6a- CYP93A1 monooxygenase 1.14.13.30leukotriene-B₄ 20-monooxygenase CYP4F 1.14.13.37methyltetrahydroprotoberberine 14- CYP93A1 monooxygenase 1.14.13.41tyrosine N-monooxygenase CYP79 1.14.13.42 hydroxyphenylacetonitrile 2- —monooxygenase 1.14.13.47 (−)-limonene 3-monooxygenase — 1.14.13.48(−)-limonene 6-monooxygenase — 1.14.13.49 (−)-limonene 7-monooxygenase —1.14.13.52 isoflavone 3′-hydroxylase — 1.14.13.53 isoflavone2′-hydroxylase — 1.14.13.55 protopine 6-monooxygenase — 1.14.13.56dihydrosanguinarine 10-monooxygenase — 1.14.13.57 dihydrochelirubine12-monooxygenase — 1.14.13.60 27-hydroxycholesterol 7α-monooxygenase —1.14.13.70 sterol 14-demethylase CYP51 1.14.13.71 N-methylcoclaurine3′-monooxygenase CYP80B1 1.14.13.73 tabersonine 16-hydroxylase CYP71D121.14.13.74 7-deoxyloganin 7-hydroxylase — 1.14.13.75 vinorinehydroxylase — 1.14.13.76 taxane 10β-hydroxylase CYP725A1 1.14.13.77taxane 13α-hydroxylase CYP725A2 1.14.13.78 ent-kaurene oxidase CYP7011.14.13.79 ent-kaurenoic acid oxidase CYP88A 1.14.14.1 unspecificmonooxygenase multiple 1.14.15.1 camphor 5-monooxygenase CYP1011.14.15.3 alkane 1-monooxygenase CYP4A 1.14.15.4 steroid11β-monooxygenase CYP11B 1.14.15.5 corticosterone 18-monooxygenaseCYP11B 1.14.15.6 cholesterol monooxygenase (side-chain- CYP11A cleaving)1.14.21.1 (S)-stylopine synthase — 1.14.21.2 (S)-cheilanthifolinesynthase — 1.14.21.3 berbamunine synthase CYP80 1.14.21.4 salutaridinesynthase — 1.14.21.5 (S)-canadine synthase — 1.14.99.9 steroid17α-monooxygenase CYP17 1.14.99.10 steroid 21-monooxygenase CYP211.14.99.22 ecdysone 20-monooxygenase — 1.14.99.28 linalool8-monooxygenase CYP111 4.2.1.92 hydroperoxide dehydratase CYP74 5.3.99.4prostaglandin-I synthase CYP8 5.3.99.5 thromboxane-A synthase CYP5

In some embodiments, the heme enzyme variant comprises a mutation at theaxial position of the heme coordination site. In some instances, themutation is an amino acid substitution of the naturally occuring residueat this position with Ala, Asp, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile,Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val at the axialposition. In other instances, the mutation is an amino acid substitutionof Cys with Asp or Ser at the axial position.

In some embodiments, the engineered heme enzyme is expressed in abacterial, archaeal or fungal host organism.

In some embodiments, the cytochrome P450 enzyme is a P450 BM3 enzyme ora variant thereof. In some instances, the P450 BM3 enzyme comprises theamino acid sequence set forth in SEQ ID NO: 1 or a variant thereof.

In some embodiments, the P450 enzyme variant comprises a mutation at theaxial position of the heme coordination site. In some instances, themutation is an amino acid substitution of Cys with Ala, Asp, Arg, Asn,Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr orVal at the axial position. In other instances, the mutation is an aminoacid substitution of Cys with Asp or Ser at the axial position.

In some embodiments, the P450BM3 enzyme comprises at least one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve, or allthirteen of the following amino acid substitutions in SEQ ID NO:1: V78A,F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V,I366V, and E442K.

In some embodiments, the cytochrome P450BM3 enzyme variant comprises aT268A mutation and/or a C400X mutation in SEQ ID NO:1, wherein X is anyamino acid other than Cys. In another embodiment, the cytochrome P450BM3enzyme variant comprises a T438S mutation and/or a C400X mutation in SEQID NO:1, wherein X is any amino acid other than Cys.

In one embodiment, the heme enzyme variant for use in the catalysis of anitrene insertion into a —S— bond to produce a product having a new S—Nbond is a P450BM3 variant comprising the following amino acidsubstitutions to SEQ ID NO: 1: V78A, F87V, P142S, T175I, A184V, S226R,H236Q, E252G, T268A, A290V, L353V, I366V, and E442K. In anotherembodiment, the heme variant optionally comprises the followingadditional amino acid substitutions to SEQ ID NO:1: L75A, I263A andL437A. In yet another embodiment, the heme variant optionally comprisesthe additional amino acid substitution C400S to SEQ ID NO:1. In someembodiments, the heme enzyme variant is the H2-5-F10 variant (see, Table7). In other embodiments, the heme enzyme variant is the P411-CISvariant (see, Table 4).

Table 3 below lists additional cyctochrome P450 enzymes that aresuitable for use in the sulfimidation or sulfoximidation reactions ofthe disclosure. The accession numbers in Table 3 are incorporated hereinby reference in their entirety for all purposes. The cytochrome P450gene and/or protein sequences disclosed in the following patentdocuments are hereby incorporated by reference in their entirety for allpurposes: WO 2013/076258; CN103160521; CN 103223219; KR 2013081394; JP5222410; WO 2013/073775; WO 2013/054890; WO 2013/048898; WO 2013/031975;WO 2013/064411; U.S. Pat. No. 8,361,769; WO 2012/150326, CN 102747053;CN 102747052; JP 2012170409; WO 2013/115484; CN 103223219; KR2013081394; CN 103194461; JP 5222410; WO 2013/086499; WO 2013/076258; WO2013/073775; WO 2013/064411; WO 2013/054890; WO 2013/031975; U.S. Pat.No. 8,361,769; WO 2012/156976; WO 2012/150326; CN 102747053; CN102747052; US 20120258938; JP 2012170409; CN 102399796; JP 2012055274;WO 2012/029914; WO 2012/028709; WO 2011/154523; JP 2011234631; WO2011/121456; EP 2366782; WO 2011/105241; CN 102154234; WO 2011/093185;WO 2011/093187; WO 2011/093186; DE 102010000168; CN 102115757; CN102093984; CN 102080069; JP 2011103864; WO 2011/042143; WO 2011/038313;JP 2011055721; WO 2011/025203; JP 2011024534; WO 2011/008231; WO2011/008232; WO 2011/005786; IN 2009DE01216; DE 102009025996; WO2010/134096; JP 2010233523; JP 2010220609; WO 2010/095721; WO2010/064764; US 20100136595; JP 2010051174; WO 2010/024437; WO2010/011882; WO 2009/108388; US 20090209010; US 20090124515; WO2009/041470; KR 2009028942; WO 2009/039487; WO 2009/020231; JP2009005687; CN 101333520; CN 101333521; US 20080248545; JP 2008237110;CN 101275141; WO 2008/118545; WO 2008/115844; CN 101255408; CN101250506; CN 101250505; WO 2008/098198; WO 2008/096695; WO 2008/071673;WO 2008/073498; WO 2008/065370; WO 2008/067070; JP 2008127301; JP2008054644; KR 794395; EP 1881066; WO 2007/147827; CN 101078014; JP2007300852; WO 2007/048235; WO 2007/044688; WO 2007/032540; CN 1900286;CN 1900285; JP 2006340611; WO 2006/126723; KR 2006029792; KR 2006029795;WO 2006/105082; WO 2006/076094; US 2006/0156430; WO 2006/065126; JP2006129836; CN 1746293; WO 2006/029398; JP 2006034215; JP 2006034214; WO2006/009334; WO 2005/111216; WO 2005/080572; US 2005/0150002; WO2005/061699; WO 2005/052152; WO 2005/038033; WO 2005/038018; WO2005/030944; JP 2005065618; WO 2005/017106; WO 2005/017105; US20050037411; WO 2005/010166; JP 2005021106; JP 2005021104; JP2005021105; WO 2004/113527; CN 1472323; JP 2004261121; WO 2004/013339;WO 2004/011648; DE 10234126; WO 2004/003190; WO 2003/087381; WO2003/078577; US 20030170627; US 20030166176; US 20030150025; WO2003/057830; WO 2003/052050; CN 1358756; US 20030092658; US 20030078404;US 20030066103; WO 2003/014341; US 20030022334; WO 2003/008563; EP1270722; US 20020187538; WO 2002/092801; WO 2002/088341; US 20020160950;WO 2002/083868; US 20020142379; WO 2002/072758; WO 2002/064765; US20020076777; US 20020076774; US 20020076774; WO 2002/046386; WO2002/044213; US 20020061566; CN 1315335; WO 2002/034922; WO 2002/033057;WO 2002/029018; WO 2002/018558; JP 2002058490; US 20020022254; WO2002/008269; WO 2001/098461; WO 2001/081585; WO 2001/051622; WO2001/034780; CN 1271005; WO 2001/011071; WO 2001/007630; WO 2001/007574;WO 2000/078973; U.S. Pat. No. 6,130,077; JP 2000152788; WO 2000/031273;WO 2000/020566; WO 2000/000585; DE 19826821; JP 11235174; U.S. Pat. No.5,939,318; WO 99/19493; WO 99/18224; U.S. Pat. No. 5,886,157; WO99/08812; U.S. Pat. No. 5,869,283; JP 10262665; WO 98/40470; EP 776974;DE 19507546; GB 2294692; U.S. Pat. No. 5,516,674; JP 07147975; WO94/29434; JP 06205685; JP 05292959; JP 04144680; DD 298820; EP 477961;SU 1693043; JP 01047375; EP 281245; JP 62104583; JP 63044888; JP62236485; JP 62104582; and JP 62019084.

TABLE 3 Additional cytochrome P450 enzymes of the disclosure. SEQSpecies Cyp No. Accession No. ID NO Bacillus megaterium 102A1 AAA87602 1Bacillus megaterium 102A1 ADA57069 2 Bacillus megaterium 102A1 ADA570683 Bacillus megaterium 102A1 ADA57062 4 Bacillus megaterium 102A1ADA57061 5 Bacillus megaterium 102A1 ADA57059 6 Bacillus megaterium102A1 ADA57058 7 Bacillus megaterium 102A1 ADA57055 8 Bacillusmegaterium 102A1 ACZ37122 9 Bacillus megaterium 102A1 ADA57057 10Bacillus megaterium 102A1 ADA57056 11 Mycobacterium sp. HXN-1500 153A6CAH04396 12 Tetrahymena thermophile 5013C2 ABY59989 13 Nonomuraeadietziae AGE14547.1 14 Homo sapiens 2R1 NP_078790 15 Macca mulatta 2R1NP_001180887.1 16 Canis familiaris 2R1 XP_854533 17 Mus musculus 2R1AAI08963 18 Bacillus halodurans C-125 152A6 NP_242623 19 Streptomycesparvus aryC AFM80022 20 Pseudomonas putida 101A1 P00183 21 Homo sapiens2D7 AAO49806 22 Rattus norvegicus C27 AAB02287 23 Oryctolagus cuniculus2B4 AAA65840 24 Bacillus subtilis 102A2 O08394 25 Bacillus subtilis102A3 O08336 26 B. megaterium DSM 32 102A1 P14779 27 B. cereus ATCC14579102A5 AAP10153 28 B. licheniformis ATTC1458 102A7 YP 079990 29 B.thuringiensis serovar X YP 037304 30 konkukian str.97-27 R.metallidurans CH34 102E1 YP 585608 31 A. fumigatus Af293 505X EAL9266032 A. nidulans FGSC A4 505A8 EAA58234 33 A. oryzae ATCC42149 505A3Q2U4F1 34 A. oryzae ATCC42149 X Q2UNA2 35 F. oxysporum 505A1 Q9Y8G7 36G. moniliformis X AAG27132 37 G. zeae PH1 505A7 EAA67736 38 G. zeae PH1505C2 EAA77183 39 M. grisea 70-15 syn 505A5 XP 365223 40 N. crassa OR74A 505A2 XP 961848 41 Oryza sativa* 97A Oryza sativa* 97B Oryza sativa97C ABB47954 42 Note: the start methionine (“M”) may be present orabsent from these sequences.

In some embodiments, the heme enzyme variant comprises a fragment of thecytochrome P450 enzyme or variant thereof. In some embodiments, the hemeenzyme variant is a cytochrome P450 BM3 enzyme variant selected fromTable 4, Table 5 and Table 6.

TABLE 4 Examplary cytochrome P450BM3 enzymes variants of the disclosure.P450_(BM3) variants Mutation compared to wild-type P450_(BM3) (SEQ IDNO: 1) P450_(BM3) (WT-BM3; SEQ ID NO: 1) None P450_(BM3)-C400A(WT-C400A) C400A P450_(BM3)-T268A (BM3-T268) T268A P411_(BM3) (ABC)C400S P411_(BM3)-T268A (ABC-T268A) T268A, C400S P411_(BM3)-T438S(ABC-T438A) T438S, C400S 9-10A R47C, V78A, K94I, P142S, T175I, A184V,F205C, S226R, H236Q, E252G, R255S, A290V, L353V B1SYN 9-10A + C47S,N70Y, A78L, F87A, I174N, I94K, V184T, I263M, G315S, A330V 9-10A TS V78A,P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K9-10A-TS-F87V 9-10A TS + F87V H2A10 9-10A TS + F87V, L75A, L181A, T268AH2-5-F10 9-10A TS + F87V, L75A, I263A, T268A, L437A H2-4-D4 9-10A TS +F87V, L75A, M177A, L181A, T268A, L437A BM3-CIS (P450_(BM3)-CIS; C3C)9-10A TS + F87V, T268A BM3-CIS-I263A BM3-CIS + I263A BM3-CIS-A328GBM3-CIS + A328G BM3-CIS-T438S BM3-CIS + T438S BM3-CIS-C400S(P411_(BM3)-CIS; ABC-CIS) BM3-CIS + C400S BM3-CIS-C400S-A268T(P411_(BM3)-CIS; BM3-CIS + C400S + A268T (9-10A TS + P87V, C400S)ABC-CIS-A268T) BM3-CIS-C400D (BM3-CIS-AxD) BM3-CIS + C400D BM3-CIS-C400Y(BM3-CIS-AxY) BM3-CIS + C400Y BM3-CIS-C400K (BM3-CIS-AxK) BM3-CIS +C400K BM3-CIS-C400H (BM3-CIS-AxH) BM3-CIS + C400H BM3-CIS-C400M(BM3-CIS-AxM) BM3-CIS + C400M WT-BM3 (heme) WT heme domain (amino acids1-463 of SEQ ID NO: 1) WT-AxA (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400A WT-AxD (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400D WT-AxH (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400H WT-AxK (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400K WT-AxM (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400M WT-AxN (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400N WT-AxS (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400S WT-AxY (heme) WT heme domain (amino acids 1-463of SEQ ID NO: 1) + C400Y BM3-CIS-T438S-AxA BM3-CIS-T438S + C400ABM3-CIS-T438S-AxD BM3-CIS-T438S + C400D BM3-CIS-T438S-AxMBM3-CIS-T438S + C400M BM3-CIS-T438S-AxY BM3-CIS-T438S + C400YBM3-CIS-T438S-AxT BM3-CIS-T438S + C400T 7-11D R47C, V78A, K94I, P142S,T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, A82F,A328V

One skilled in the art will understand that any of the mutations listedin Table 4 can be introduced into any cytochrome P450 enzyme of interestby locating the segment of the DNA sequence in the correspondingcytochrome P450 gene which encodes the conserved amino acid residue asdescribed above for identifying the conserved cysteine residue in acytochrome P450 enzyme of interest that serves as the heme axial ligand.In certain instances, this DNA segment is identified through detailedmutagenesis studies in a conserved region of the protein (see, e.g.,Shimizu et al., Biochemistry 27, 4138-4141, 1988). In other instances,the conserved amino acid residue is identified through crystallographicstudy (see, e.g., Poulos et al., J. Mol. Biol 195:687-700, 1987). In yetother instances, protein sequence alignment algorithms can be used toidentify the conserved amino acid residue. For example, BLAST alignmentwith the P450 BM3 amino acid sequence as the query sequence can be usedto identify the heme axial ligand site and/or the equivalent T268residue in other cytochrome P450 enzymes.

Table 5 below provides non-limiting examples of cytochrome P450 BM3variants of the disclosure. Each P450 BM3 variant comprises one or moreof the listed mutations (Variant Nos. 1-31), wherein a “+” indicates thepresence of that particular mutation in the variant. Any of the variantslisted in Table 4 can further comprise an 1263 A and/or an A328Gmutation and/or at least one, two, three, four, or five of the followingalanine substitutions, in any combination, in the P450 BM3 enzyme activesite: L75A, M177A, L181A, I263A, and L437A. In particular embodiments,the P450 BM3 variant comprises or consists of the heme domain of any oneof Variant Nos. 1-31 listed in Table 5 or a fragment thereof, whereinthe fragment is capable of carrying out the nitrenetransfer/sulfimidation of the disclosure.

TABLE 5 Exemplary cytochrome P450 BM3 enzyme variants of the disclosure.P450_(BM3) variant Mutation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16C400X + + + + + + T268A + + + + + + F87V + + + + + + 9-10A-TS + + + + +T438Z + + + + + P450_(BM3) variant Mutation 17 18 19 20 21 22 23 24 2526 27 28 29 30 31 C400X + + + + + + + + + + T268A + + + + + + + + + +F87V + + + + + + + + + + 9-10A-TS + + + + + + + + + + +T438Z + + + + + + + + + + +Mutations relative to the wild-type P450_(BM3) amino acid sequence (SEQID NO: 1); “X” is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His,He, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; “Z” isselected from Ala, Ser, and Pro; “9-10A-TS” includes the following aminoacid substitutions in SEQ ID NO: 1: V78A, P142S, T175I, A184V, S226R,H236Q, E252G, A290V, L353V, I366V, and E442K.

In other aspects, the disclosure provides chimeric heme enzymes such as,e.g., chimeric P450 polypeptides comprised of recombined sequences fromP450 BM3 and at least two, or more distantly related P450 enzymes fromBacillus subtillis or variants. As a non-limiting example, site-directedrecombination of three bacterial cytochrome P450s can be performed withsequence crossover sites selected to minimize the number of disruptedcontacts within the protein structure. In some embodiments, sevencrossover sites can be chosen, resulting in eight sequence blocks. Oneskilled in the art will understand that the number of crossover sitescan be chosen to produce the desired number of sequence blocks, e.g., 1,2, 3, 4, 5, 6, 7, 8, or 9 crossover sites for 2, 3, 4, 5, 6, 7, 8, 9, or10 sequence blocks, respectively. In other embodiments, the numberingused for the chimeric P450 refers to the identity of the parent sequenceat each block. For example, “12312312” refers to a sequence containingblock 1 from P450 #1, block 2 from P450 #2, block 3 from P450 #3, block4 from P450 #1, block 5 from P450 #2, and so on. A chimeric libraryuseful for generating the chimeric heme enzymes of the invention can beconstructed as described in U.S. Pat. Publ. No. US-2012-0171693-A1 toArnold et al., the disclosure of which is incorporated herein for allpurposes.

As a non-limiting example, chimeric P450 proteins comprising recombinedsequences or blocks of amino acids from CYP102A1 (Accession No. J04832),CYP102A2 (Accession No. CAB12544), and CYP102A3 (Accession No. U93874)can be constructed. In certain instances, the CYP102A1 parent sequenceis assigned “1”, the CYP102A2 parent sequence is assigned “2”, and theCYP102A3 is parent sequence assigned “3”. In some instances, each parentsequence is divided into eight sequence blocks containing the followingamino acids (aa): block 1: aa 1-64; block 2: aa 65-122; block 3: aa123-166; block 4: aa 167-216; block 5: aa 217-268; block 6: aa 269-328;block 7: aa 329-404; and block 8: aa 405-end. Thus, in this example,there are eight blocks of amino acids and three fragments are possibleat each block. For instance, “12312312” refers to a chimeric P450protein of the invention containing block 1 (aa 1-64) from CYP102A1,block 2 (aa 65-122) from CYP102A2, block 3 (aa 123-166) from CYP102A3,block 4 (aa 167-216) from CYP102A1, block 5 (aa 217-268) from CYP102A2,and so on. Non-limiting examples of chimeric P450 proteins include thoseset forth in Table 6 (C2G9, X7, X7-12, C2E6, X7-9, C2B12, TSP234). Insome embodiments, the chimeric heme enzymes of the invention cancomprise at least one or more of the mutations described herein.

TABLE 6 Exemplary preferred chimeric cytochrome P450 enzymes of theinvention. Chimeric P450s Heme domain block sequence SEQ ID NO C2G922223132 43 X7 22312333 44 X7-12 12112333 45 C2E6 11113311 46 X7-932312333 47 C2B12 32313233 48 TSP234 22313333 49

In another embodiment, the disclosure provides a method for catalyzing anitrene insertion or transfer into a —S— bond to produce a product witha new S—N bond. The method comprising: providing a —S— containingsubstrate, a nitrene precursor and an engineered P450 enzyme asdescribed herein and above; and allowing the reaction to proceed for atime sufficient to form a product having a new S—N bond.

In some embodiments, the engineered P450 enzyme is expressed in abacterial, archaeal or fungal host organism.

In some embodiments, the cytochrome P450 enzyme is a P450 BM3 enzyme ora variant thereof. In some instances, the P450 BM3 enzyme comprises theamino acid sequence set forth in SEQ ID NO: 1 or a variant thereof.

An enzyme's total turnover number (or TTN) refers to the maximum numberof molecules of a substrate that the enzyme can convert before becominginactivated. In general, the TTN for the heme enzymes of the disclosurerange from about 1 to about 100,000 or higher. For example, the TTN canbe from about 1 to about 1,000, or from about 1,000 to about 10,000, orfrom about 10,000 to about 100,000, or from about 50,000 to about100,000, or at least about 100,000. In particular embodiments, the TTNcan be from about 100 to about 10,000, or from about 10,000 to about50,000, or from about 5,000 to about 10,000, or from about 1,000 toabout 5,000, or from about 100 to about 1,000, or from about 250 toabout 1,000, or from about 100 to about 500, or at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000,20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000,65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, ormore. In certain embodiments, the variant or chimeric heme enzymes ofthe disclosure have higher TTNs compared to the wild-type sequences. Insome instances, the variant or chimeric heme enzymes have TTNs greaterthan about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350,400, 450, 500, or more) in carrying out in vitro sulfimidationreactions. In other instances, the variant or chimeric heme enzymes haveTTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000,10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out invivo whole cell reactions.

In general, the term “mutant” or “variant” as used herein with referenceto a molecule such as polynucleotide or polypeptide, indicates that hasbeen mutated from the molecule as it exits in nature. In particular, theterm “mutate” and “mutation” as used herein indicates any modificationof a nucleic acid and/or polypeptide which results in an altered nucleicacid or polypeptide. Mutations include any process or mechanismresulting in a mutant protein, enzyme, polynucleotide, gene, or cell.This includes any mutation in which a polynucleotide or polypeptidesequence is altered, as well as any detectable change in a cell whereinthe mutant polynucleotide or polypeptide is expressed arising from sucha mutation. Typically, a mutation occurs in a polynucleotide or genesequence, by point mutations, deletions, or insertions of single ormultiple nucleotide residues. A mutation in a polynucleotide includesmutations arising within a protein-encoding region of a gene as well asmutations in regions outside of a protein-encoding sequence, such as,but not limited to, regulatory or promoter sequences. A mutation in acoding polynucleotide such as a gene can be “silent”, i.e., notreflected in an amino acid alteration upon expression, leading to a“sequence-conservative” variant of the gene. A mutation in a polypeptideincludes but is not limited to mutation in the polypeptide sequence andmutation resulting in a modified amino acid. Non-limiting examples of amodified amino acid include a glycosylated amino acid, a sulfated aminoacid, a prenylated (e.g., farnesylated, geranylgeranylated) amino acid,an acetylated amino acid, an acylated amino acid, a PEGylated aminoacid, a biotinylated amino acid, a carboxylated amino acid, aphosphorylated amino acid, and the like. References adequate to guideone of skill in the modification of amino acids are replete throughoutthe literature. Example protocols are found in Walker (1998) ProteinProtocols on CD-ROM (Humana Press, Towata, N.J.).

A mutant or engineered protein or enzyme is usually, although notnecessarily, expressed from a mutant polynucleotide or gene. Engineeredcells can be obtained by introduction of an engineered gene or part ofit in the cell. The terms “engineered cell”, “mutant cell” or“recombinant cell” as used herein refer to a cell that has been alteredor derived, or is in some way different or changed, from a parent cell,including a wild-type cell. The term “recombinant” as used herein withreference to a cell in alternative to “wild-type” or “native”, indicatesa cell that has been engineered to modify the genotype and/or thephenotype of the cell as found in nature, e.g., by modifying thepolynucleotides and/or polypeptides expressed in the cell as it existsin nature. A “wild-type cell” refers instead to a cell which has notbeen engineered and displays the genotype and phenotype of said cell asfound in nature.

The term “engineer” refers to any manipulation of a molecule or cellthat result in a detectable change in the molecule or cell, wherein themanipulation includes but is not limited to inserting a polynucleotideand/or polypeptide heterologous to the cell and mutating apolynucleotide and/or polypeptide native to the cell. Engineered cellscan also be obtained by modification of the cell' genetic material,lipid distribution, or protein content. In addition to recombinantproduction, the enzymes may be produced by direct peptide synthesisusing solid-phase techniques, such as Solid-Phase Peptide Synthesis.Peptide synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.) in accordance with the instructions provided by the manufacturer

Variants of naturally-occurring sequences can be generated bysite-directed mutagenesis (Botstein and Shortie 1985; Smith 1985; Carter1986; Dale and Felix 1996; Ling and Robinson 1997), mutagenesis usinguracil containing templates (Kunkel, Roberts et al. 1987; Bass, Sorrellset al. 1988), oligonucleotide-directed mutagenesis (Zoller and Smith1983; Zoller and Smith 1987; Zoller 1992), phosphorothioate-modified DNAmutagenesis (Taylor, Schmidt et al. 1985; Nakamaye and Eckstein 1986;Sayers, Schmidt et al. 1988), mutagenesis using gapped duplex DNA(Kramer, Drutsa et al. 1984; Kramer and Fritz 1987), point mismatch,mutagenesis using repair-deficient host strains, deletion mutagenesis(Eghtedarzadeh and Henikoff 1986), restriction-selection andrestriction-purification (Braxton and Wells 1991), mutagenesis by totalgene synthesis (Nambiar, Stackhouse et al. 1984; Grundstrom, Zenke etal. 1985; Wells, Vasser et al. 1985)], double-strand break repair(Mandecki 1986), and the like. Additional details on many of the abovemethods can be found in Methods in Enzymology Volume 154, which alsodescribes useful controls for trouble-shooting problems with variousmutagenesis methods.

Additional details regarding the methods to generate variants ofnaturally-occurring sequences can be found in the following U.S.patents, PCT publications, and EPO publications: U.S. Pat. No. 5,605,793to Stemmer (Feb. 25, 1997), “Methods for In vitro Recombination;” U.S.Pat. No. 5,811,238 to Stemmer et al. (Sep. 22, 1998) “Methods forGenerating Polynucleotides having Desired Characteristics by IterativeSelection and Recombination;” U.S. Pat. No. 5,830,721 to Stemmer et al.(Nov. 3, 1998), “DNA Mutagenesis by Random Fragmentation andReassembly;” U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998)“End-Complementary Polymerase Reaction;” U.S. Pat. No. 5,837,458 toMinshull, et al. (Nov. 17, 1998), “Methods and Compositions for Cellularand Metabolic Engineering;” WO 95/22625, Stemmer and Crameri,“Mutagenesis by Random Fragmentation and Reassembly;” WO 96/33207 byStemmer and Lipschutz “End Complementary Polymerase Chain Reaction;” WO97/20078 by Stemmer and Crameri “Methods for Generating Polynucleotideshaving Desired Characteristics by Iterative Selection andRecombination;” WO 97/35966 by Minshull and Stemmer, “Methods andCompositions for Cellular and Metabolic Engineering;” WO 99/41402 byPunnonen et al. “Targeting of Genetic Vaccine Vectors;” WO 99/41383 byPunnonen et al. “Antigen Library Immunization;” WO 99/41369 by Punnonenet al. “Genetic Vaccine Vector Engineering;” WO 99/41368 by Punnonen etal. “Optimization of Immunomodulatory Properties of Genetic Vaccines;”EP 752008 by Stemmer and Crameri, “DNA Mutagenesis by RandomFragmentation and Reassembly;” EP 0932670 by Stemmer “Evolving CellularDNA Uptake by Recursive Sequence Recombination;” WO 99/23107 by Stemmeret al., “Modification of Virus Tropism and Host Range by Viral GenomeShuffling;” WO 99/21979 by Apt et al., “Human Papillomavirus Vectors;”WO 98/31837 by del Cardayre et al. “Evolution of Whole Cells andOrganisms by Recursive Sequence Recombination;” WO 98/27230 by Pattenand Stemmer, “Methods and Compositions for Polypeptide Engineering;” WO98/13487 by Stemmer et al., “Methods for Optimization of Gene Therapy byRecursive Sequence Shuffling and Selection;” WO 00/00632, “Methods forGenerating Highly Diverse Libraries;” WO 00/09679, “Methods forObtaining in vitro Recombined Polynucleotide Sequence Banks andResulting Sequences;” WO 98/42832 by Arnold et al., “Recombination ofPolynucleotide Sequences Using Random or Defined Primers;” WO 99/29902by Arnold et al., “Method for Creating Polynucleotide and PolypeptideSequences;” WO 98/41653 by Vind, “An in vitro Method for Construction ofa DNA Library;” WO 98/41622 by Borchert et al., “Method for Constructinga Library Using DNA Shuffling;” WO 98/42727 by Pati and Zarling,“Sequence Alterations using Homologous Recombination;” WO 00/18906 byPatten et al., “Shuffling of Codon-Altered Genes;” WO 00/04190 by delCardayre et al. “Evolution of Whole Cells and Organisms by RecursiveRecombination;” WO 00/42561 by Crameri et al., “Oligonucleotide MediatedNucleic Acid Recombination;” WO 00/42559 by Selifonov and Stemmer“Methods of Populating Data Structures for Use in EvolutionarySimulations;” WO 00/42560 by Selifonov et al., “Methods for MakingCharacter Strings, Polynucleotides & Polypeptides Having DesiredCharacteristics;” WO 01/23401 by Welch et al., “Use of Codon-VariedOligonucleotide Synthesis for Synthetic Shuffling;” and WO 01/64864“Single-Stranded Nucleic Acid Template-Mediated Recombination andNucleic Acid Fragment Isolation” by Affholter.

In particular, in some embodiments, site-directed mutagenesis can beperformed on predetermined residues of a heme containing enzyme or P450polypeptide. These predetermined sites can be identified using thecrystal structure of the heme or P450 enzyme if available or a crystalstructure of a homologous protein that shares at least 20% sequenceidentity with a heme or P450 enzyme of the disclosure and an alignmentof the polynucleotide or amino acid sequences and its homologousprotein. Mutagenesis of the predetermined sites can be performedchanging one, two or three of the nucleotides in the codon that encodesfor each of the predetermined amino acids. Mutagenesis of thepredetermined sites can be performed in the described way so that eachof the predetermined amino acid is mutated to any of the other 19natural amino acids. Substitution of the predetermined sites withunnatural amino acids can be performed using methods established in vivo(Wang, Xie et al. 2006), in vitro (Shimizu, Kuruma et al. 2006),semisynthetic (Schwarzer and Cole 2005) or synthetic methods (Camareroand Mitchell 2005) for incorporation of unnatural amino acids intopolypeptides.

In still further embodiments, libraries of engineered variants can beobtained by laboratory evolutionary methods and/or rational designmethods, using one or a combination of techniques such as randommutagenesis, site-saturation mutagenesis, site-directed mutagenesis, DNAshuffling, DNA recombination, and the like and targeting one or more ofthe amino acid residues, one at a time or simultaneously. Said librariescan be arrayed on multi-well plates and screened for activity on thetarget molecule using a colorimetric, fluorimetric, enzymatic, orluminescence assay and the like. For example a method for makinglibraries for directed evolution to obtain P450s with new or alteredproperties is recombination, or chimeragenesis, in which portions ofhomologous P450s are swapped to form functional chimeras, can use used.Recombining equivalent segments of homologous proteins generatesvariants in which every amino acid substitution has already proven to besuccessful in one of the parents. Therefore, the amino acid mutationsmade in this way are less disruptive, on average, than random mutations.A structure-based algorithm, such as SCHEMA, can be used to identifyfragments of proteins that can be recombined to minimize disruptiveinteractions that would prevent the protein from folding into its activeform.

In some embodiments, activation of a target site in an organic moleculecan be performed in a whole-cell system. To prepare the whole-cellsystem, the encoding sequence can be introduced into a host cell using asuitable vector, such as a plasmid, a cosmid, a phage, a virus, abacterial artificial chromosome (BAC), a yeast artificial chromosome(YAC), or the like, into which the said sequence of the disclosure hasbeen inserted, in a forward or reverse orientation. In some embodiments,the construct further comprises regulatory sequences, including, forexample, a promoter linked to the sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art, and arecommercially available.

Accordingly, in other embodiments, vectors that include a nucleic acidmolecule of the disclosure are provided. In other embodiments, hostcells transfected with a nucleic acid molecule of the disclosure, or avector that includes a nucleic acid molecule of the disclosure, areprovided. Host cells include eucaryotic cells such as yeast cells,insect cells, or animal cells. Host cells also include procaryotic cellssuch as bacterial cells.

In other embodiments, methods for producing a cell for carrying out orproducing an enzyme catalyst of the disclosure are provided. Suchmethods generally include: (a) transforming a cell with an isolatednucleic acid molecule encoding a polypeptide having the enzymaticactivity that transfers or inserts a nitrene into a —S— target site; (b)transforming a cell with an isolated nucleic acid molecule encoding apolypeptide of the disclosure; or (c) transforming a cell with anisolated nucleic acid molecule of the disclosure.

The terms “vector”, “vector construct” and “expression vector” as usedherein refer to a vehicle by which a DNA or RNA sequence (e.g. a foreigngene) can be introduced into a host cell, so as to transform the hostand promote expression (e.g. transcription and translation) of theintroduced sequence. Vectors typically comprise the DNA of atransmissible agent, into which foreign DNA encoding a protein isinserted by restriction enzyme technology. A common type of vector is a“plasmid”, which generally is a self-contained molecule ofdouble-stranded DNA that can readily accept additional (foreign) DNA andwhich can readily introduced into a suitable host cell. A large numberof vectors, including plasmid and fungal vectors, have been describedfor replication and/or expression in a variety of eukaryotic andprokaryotic hosts. Non-limiting examples include pKK plasmids(Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.),pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids(New England Biolabs, Beverly, Mass.), and many appropriate host cells,using methods disclosed or cited herein or otherwise known to thoseskilled in the relevant art. Recombinant cloning vectors will ofteninclude one or more replication systems for cloning or expression, oneor more markers for selection in the host, e.g., antibiotic resistance,and one or more expression cassettes.

The terms “express” and “expression” refers to allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Apolynucleotide or polypeptide is expressed recombinantly, for example,when it is expressed or produced in a foreign host cell under thecontrol of a foreign or native promoter, or in a native host cell underthe control of a foreign promoter.

Polynucleotides provided herein can be incorporated into any one of avariety of expression vectors suitable for expressing a polypeptide.Suitable vectors include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, pseudorabies, adenovirus, adeno-associated viruses, retrovirusesand many others. Any vector that transduces genetic material into acell, and, if replication is desired, which is replicable and viable inthe relevant host can be used.

Vectors can be employed to transform an appropriate host to permit thehost to express a protein or polypeptide. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli, B. subtilis,Streptomyces, and Salmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insectcells such as Drosophila and Spodoptera frugiperda; mammalian cells suchas CHO, COS, BHK, HEK 293 br Bowes melanoma; or plant cells or explants,etc.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the polypeptide. For example, suchvectors include, but are not limited to, multifunctional E. coli cloningand expression vectors such as BLUESCRIPT (Stratagene), in which thecoding sequence may be ligated into the vector in-frame with sequencesfor the amino-terminal Met and the subsequent 7 residues ofbeta-galactosidase so that a hybrid protein is produced; pIN vectors;pET vectors; and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used for production of an enzyme catalystof the disclosure.

In order to perform the sulfimidation reactions described herein, anitrene precursor is used. The nitrene precursor can be an azide. Forexample, the nitrene precursor can have the general formula: R¹—N₃,wherein R¹ is:

(i) a substituted or unsubstituted aryl, a substitute or unsubstitutedalkyl, —OR², or —NR², wherein R² is a substituted or unsubstituted aryl,a substitute or unsubstituted alkyl;

(ii) —SO₂R³, wherein R³ a substituted or unsubstituted aryl, asubstitute or unsubstituted alkyl, —OR² or NR², wherein R² are any alkylor aryl;

(iii) —COR⁴ wherein R⁴ is a substituted or unsubstituted aryl, asubstitute or unsubstituted alkyl, —OR² or NR², wherein R² are any alkylor aryl; or

(iv) —P(O)(OR⁵)(OR⁶), wherein R⁵ and R⁶ are independently H, asubstituted or unsubstituted aryl, a substitute or unsubstituted alkyl.

In another embodiment, the azide has a structure selected from the groupconsisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl. In a specific embodiment, the azide is a tosyl azide.

The nitrene precursor can also be is selected from the group consistingof:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl and wherein —OTs can be ITs. In certain aspects, thenitrene precursor contains a leaving group. Suitable leaving groupsinclude, but are not limited to, OTs (tosylates), OMs (mesylates),halogen, N₂, H₂ and ITs (N-tosylimine).

In certain aspects, the disclosure provides methods and systems forheme-containing enzymes to catalyze nitrogen transfer to a sulfur in anorganosulfur compound, also known as sulfimidation or sulfoximidation.The reactions can be intermolecular, intramolecular and a combinationthereof. These heme containing enzymes catalyze the sulfimidation orsulfoximidation via nitrene transfer or insertion, which allows thegeneration of a new S—N bond. The reactions proceed with high regio,chemo, and/or diastereoselectivity as a result of uing a heme containingenzyme.

In one embodiment, the disclosure provides a method for catalyzing anitrene insertion or transfer to a sulfur atom targe in an oranosulfurcompound to produce a product having a new S—N bond. The methodcomprises: providing a sulfur containing substrate, a nitrene precursorand an engineered heme enzyme; and allowing the reaction to proceed fora time sufficient to form a product having a new S—N bond. In otherembodiments, the disclosure provides a product of the methods herein.

In certain embodiments, the nitrene precursor contains an azidefunctional group. In one embodiment, a product obtained from the methodsof the disclosure comprises a compound of Formula 1a:

wherein R¹ is a sulfoxide, a carbonyl or a phosphonate; wherein R² is Hor any alkyl or aryl; and wherein R³ is H, O or an optionallysubstituted aryl group. In one embodiment, R¹ is a sulfoxide of formulaSO₂R⁵, wherein R⁵ any alkyl, any aryl, —OR⁶ or NR⁷, wherein R⁶ and R⁷are any alkyl or any aryl. IN a further embodiment, R² is any alkyl oraryl. In still a further or alternative embodiment, R³ is H. In anotherembodiment, R¹ is a phosphonate of formula P(O)(OR⁸)(OR⁹), wherein R⁸and R⁹ are independently any aryl or any alkyl. In a further embodiment,R² is any alkyl or any aryl. In still a further embodiment, R³ is anyalkyl or aryl. In another embodiment, R¹ is a carbonyl group. In afurther embodiment, R² is any alkyl or any aryl. In still a furtherembodiment, R³ is any alkyl or any aryl. In another embodiment, R³ is anoptionally substituted aryl group. In a further embodiment, R¹ is anyalkyl or any aryl. In still a further embodiment, R² is H or any alkylor any aryl. In another embodiment, R¹ is a carbonyl group. In a furtherembodiment, R² is any alkyl or any aryl. In still a further embodiment,R³ is O.

The methods of the disclosure include forming reaction mixtures thatcontain the heme enzymes described herein. The heme enzymes can be, forexample, purified prior to addition to a reaction mixture or secreted bya cell present in the reaction mixture. The reaction mixture can containa cell lysate including the enzyme, as well as other proteins and othercellular materials. Alternatively, a heme enzyme can catalyze thereaction within a cell expressing the heme enzyme. Any suitable amountof heme enzyme can be used in the methods of the disclosure. In general,the reaction mixtures contain from about 0.01 mol % to about 10 mol %heme enzyme with respect to the nitrene precursor and/or substrate. Thereaction mixtures can contain, for example, from about 0.01 mol % toabout 0.1 mol % heme enzyme, or from about 0.1 mol % to about 1 mol %heme enzyme, or from about 1 mol % to about 10 mol % heme enzyme. Thereaction mixtures can contain from about 0.05 mol % to about 5 mol %heme enzyme, or from about 0.05 mol % to about 0.5 mol % heme enzyme.The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or about 1 mol % heme enzyme.

The concentration of the organosulfur substrate and nitrene precursorare typically in the range of from about 100 μM to about 1 M. Theconcentration can be, for example, from about 100 μM to about 1 mM, orabout from 1 mM to about 100 mM, or from about 100 mM to about 500 mM,or from about 500 mM to 1 M. The concentration can be from about 500 μMto about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about 50mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30mM. The concentration of organosulfur substrate and nitrene precursorcan be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or900 μM. The concentration of organosulfur substrate and nitreneprecursor can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or500 mM.

Reaction mixtures can contain additional components. As non-limitingexamples, the reaction mixtures can contain buffers (e.g.,2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS),2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate,sodium phosphate, phosphate-buffered saline, sodium citrate, sodiumacetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, isopropanol, glycerol,tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g.,NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., ureaand guandinium hydrochloride), detergents (e.g., sodium dodecylsulfateand Triton-X 100), chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid(BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducingagents (e.g., sodium dithionite, NADPH, NADH, dithiothreitol (DTT),β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)).Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars,and reducing agents can be used at any suitable concentration, which canbe readily determined by one of skill in the art. In general, buffers,cosolvents, salts, denaturants, detergents, chelators, sugars, andreducing agents, if present, are included in reaction mixtures atconcentrations ranging from about 1 μM to about 1 M. For example, abuffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, asugar, or a reducing agent can be included in a reaction mixture at aconcentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, orabout 250 mM, or about 500 mM, or about 1 M. In some embodiments, areducing agent is used in a sub-stoichiometric amount with respect tothe organosulfur substrate and nitrene precursor. Cosolvents, inparticular, can be included in the reaction mixtures in amounts rangingfrom about 1% v/v to about 75% v/v, or higher. A cosolvent can beincluded in the reaction mixture, for example, in an amount of about 5,10, 20, 30, 40, or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze theformation of the desired products. The reactions can be conducted at anysuitable temperature. In general, the reactions are conducted at atemperature of from about 4° C. to about 40° C. The reactions can beconducted, for example, at about 25° C. or about 37° C. The reactionscan be conducted at any suitable pH. In general, the reactions areconducted at a pH of from about 6 to about 10. The reactions can beconducted, for example, at a pH of from about 6.5 to about 9. Thereactions can be conducted for any suitable length of time. In general,the reaction mixtures are incubated under suitable conditions foranywhere between about 1 minute and several hours. The reactions can beconducted, for example, for about 1 minute, or about 5 minutes, or about10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, orabout 4 hours, or about 8 hours, or about 12 hours, or about 24 hours,or about 48 hours, or about 72 hours. Reactions can be conducted underaerobic conditions or anaerobic conditions. Reactions can be conductedunder an inert atmosphere, such as a nitrogen atmosphere or argonatmosphere. In some embodiments, a solvent is added to the reactionmixture. In some embodiments, the solvent forms a second phase, and thecyclopropanation occurs in the aqueous phase. In some embodiments, theheme enzyme is located in the aqueous layer whereas the substratesand/or products occur in an organic layer. Other reaction conditions maybe employed in the methods of the disclosure, depending on the identityof a particular heme enzyme, organosulfur substrate and nitreneprecursor.

Reactions can be conducted in vivo with intact cells expressing a hemeenzyme of the disclosure. The in vivo reactions can be conducted withany of the host cells used for expression of the heme enzymes, asdescribed herein. A suspension of cells can be formed in a suitablemedium supplemented with nutrients (such as mineral micronutrients,glucose and other fuel sources, and the like). Nitrene transfer orinsertion yields from reactions in vivo can be controlled, in part, bycontrolling the cell density in the reaction mixtures. Cellularsuspensions exhibiting optical densities ranging from about 0.1 to about50 at 600 nm can be used for nitrene transfer reactions. Other densitiescan be useful, depending on the cell type, specific heme enzymes, orother factors.

The methods of the disclosure can be assessed in terms of thediastereoselectivity and/or enantioselectivity of sulfimidation orsulfoximidatino reaction—that is, the extent to which the reactionproduces a particular isomer, whether a diastereomer or enantiomer. Aperfectly selective reaction produces a single isomer, such that theisomer constitutes 100% of the product. As another non-limiting example,a reaction producing a particular enantiomer constituting 90% of thetotal product can be said to be 90% enantioselective. A reactionproducing a particular diastereomer constituting 30% of the totalproduct, meanwhile, can be said to be 30%>diastereoselective.

In general, the methods of the invention include reactions that are fromabout 1% to about 99% diastereoselective. The reactions are from about1% to about 99% enantioselective. The reaction can be, for example, fromabout 10% to about 90% diastereoselective, or from about 20%> to about80%>diastereoselective, or from about 40%> to about 60%)diastereoselective, or from about 1% to about 25% diastereoselective, orfrom about 25% o to about 50% diastereoselective, or from about 50% toabout 75% diastereoselective. The reaction can be about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orabout 95% diastereoselective. The reaction can be from about 10% toabout 90% enantioselective, from about 20% to about 80%enantioselective, or from about 40% to about 60% enantioselective, orfrom about 1% to about 25% enantioselective, or from about 25% to about50% enantioselective, or from about 50% to about 75% enantioselective.The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective.Accordingly some embodiments of the disclosure provide methods whereinthe reaction is at least 30% to at least 90% diastereoselective. In someembodiments, the reaction is at least 30% to at least 90%enantioselective. As described herein, the ratios and reactants (e.g.,the type of nitrene precursor the heme variant and cofactors) can bemodified to yield a desired ratio of enantiomers.

One of skill in the art will appreciate that stereochemicalconfiguration of certain of the products herein will be determined inpart by the orientation of the product of the enzymatic step. Certain ofthe products herein will be “cis” compounds or “Z” compounds. Otherproducts will be “trans” compounds or “E” compounds.

In certain instances, two cis isomers and two trans isomers can arisefrom the reaction of an organosulfur substrate and a nitrene precursor.The two cis isomers are enantiomers with respect to one another, in thatthe structures are non-superimposable mirror images of each other.Similarly, the two trans isomers are enantiomers. One of skill in theart will appreciate that the absolute stereochemistry of a product—thatis, whether a given chiral center exhibits the right-handed “R”configuration or the left-handed “S” configuration—will depend onfactors including the structures of the particular substrate and nitreneprecursor used in the reaction, as well as the identity of the enzyme.The relative stereochemistry—that is, whether a product exhibits a cisor trans configuration—as well as for the distribution of productmixtures will also depend on such factors.

In certain instances, the product mixtures have cis:trans ratios rangingfrom about 1:99 to about 99:1. The cis:trans ratio can be, for example,from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or fromabout 1:50 to about 1:25, or from about 99:1 to about 75:1, or fromabout 75:1 to about 50:1, or from about 50:1 to about 25:1. Thecis:trans ratio can be from about 1:80 to about 1:20, or from about 1:60to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 toabout 40:1. The cis:trans ratio can be about 1:5, 1:10, 1:15, 1:20,1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80,1:85, 1:90, or about 1:95. The cis:trans ratio can be about 5:1, 10:1,15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1,75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of a product mixture can be assessed in terms of theenantiomeric excess, or “% ee,” of the mixture. The enantiomeric excessrefers to the difference in the mole fractions of two enantiomers in amixture. In certain instances, as a non-limiting example, for instance,the enantiomeric excess of the “E” or trans (R,R) and (S,S) enantiomerscan be calculated using the formula: %>eeE=[(% R,R−% s,sy(% R,R+%s,s)]×100%), wherein χ is the mole fraction for a given enantiomer. Theenantiomeric excess of the “Z” or cis enantiomers (% eez) can becalculated in the same manner.

In certain instances, product mixtures exhibit % ee values ranging fromabout 1% to about 99%, or from about −1% to about −99%. The closer agiven % ee value is to 99% (or −99%), the purer the reaction mixture is.The % ee can be, for example, from about −90% to about 90%), or fromabout −80% to about 80%, or from about −70% to about 70%, or from about−60%) to about 60%, or from about −40% to about 40%, or from about −20%to about 20%). The % ee can be from about 1% to about 99%, or from about20% to about 80%, or from about 40% to about 60%, or from about 1% toabout 25%, or from about 25% to about 50%), or from about 50% to about75%. The % ee can be from about −1% to about −99%, or from about −20% toabout −80%, or from about −40% to about −60%, or from about −1% to about−25%), or from about −25% to about −50%, or from about −50% to about−75%. The % ee can be about −99%, −95%, −90%, −85%, −80%, −75%, −70%,−65%, −60%, −55%, −50%, −45%, −40%, −35%, −30%, −25%, −20%, −15%, −10%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or about 95%. Any of these values can be % eeE values or% eez values.

Accordingly, some embodiments of the disclosure provide methods forproducing a plurality of products having a % eez of from about −90% toabout 90%. In some embodiments, the % eez is at least 90%. In someembodiments, the % eez is at least −99%. In some embodiments, the % eeEis from about −90% to about 90%. In some embodiments, the % eeE is atleast 90%. In some embodiments, the % eeE is at least −99%.

EXAMPLES

The present disclosure is further illustrated in the following examples,which are provided by way of illustration and are not intended to belimiting.

Work on intramolecular C—H amination was limited to aryl sulfonylazidesubstrates as nitrene precursors. Despite the success with thissubstrate class, research was performed to assess the influence of theR-group on the nitrenoid transfer and thus a series of substratesdisplaying a range of stereoelectronic properties that have been shownto be effective nitrene precursors in other contexts were tested (FIG.5-6).

For the thioether acceptor substrate thioanisole was chosen, which hasbeen used in enzymatic sulfoxidation by cytochrome P450s and otheroxygenase enzymes. As a catalyst, P411_(BM3)-CIS T438S, a variant ofcytochrome P450_(BM3), possessing the aforementioned C400S mutation wasused. This enzyme, which contains 14 mutations relative to wild-typeP450_(BM3) (Table A), was previously shown to be a good catalyst in theactivation of azides for intramolecular C—H insertion. Reactionconditions were similar to those reported for intramolecular C—Hamination (see, e.g., International Application Publication No. WO2014/058729, incorporated herein by reference for all purposes) underanaerobic conditions with nicotine adenine dinucleotide phosphate(NADPH) supplied as a reductant.

TABLE A Mutations present in P450 BM3 variants used in the disclosureEnzyme Mutations relative to wild-type P450_(BM3) P450_(BM3) noneP450_(BM3)-T268A T268A P411_(BM3) C400S P411_(BM3)-T268A T268A, C400SP450_(BM3)-CIS T438S V78A, F87V, P142S, T175I, A184V, S226R, H236Q,E252G, A290V, L353V, I366V, T438S, E442K P411_(BM3)-CIS T438SP450BM3-CIS C400S, T438S P411_(BM3)-CIS A268T P450BM3-CIS, A268T, C400S,T438S T438S P411_(BM3) H2-A-10 P450BM3-CIS, L75A, L181A, C400S,P411_(BM3) H2-5-F10 P450BM3-CIS, L75A, I263A, C400S, L437A P411_(BM3)H2-4-D4 P450BM3-CIS, L75A, M177A, L181A, C400S, L437A, T438S

Considering the small size of reactive oxygen species naturally producedby P450s, it was anticipated that smaller azides, such as mesyl azidewould be less sterically demanding than aryl or arylsulfonyl azides andthus a more suitable partner for reaction with thioanisole. Tosyl azidewas shown to be an exception precursor for sulfimidation.

Control experiments confirmed that enzyme was necessary for sulfimideformation (Table 7). Free hemin showed no activity in thistransformation.

In other non-natural P450 reactions reported to date, it was shown thatamino acid substitutions could alter both the activity andstereoselectivity of the enzymes. Thus, mutation of conserved residuesC400 and T268 and other active-site residues were tested to determinetheir effect on sulfimidation activity (Table B). For these experimentswe used the more reactive sulfide 4-methoxythioanisole, for which wemeasured 300 TTN with P411BM3-CIS T438S (see below for more discussionof the effect of sulfide substituents on reactivity).

TABLE B Sulfimidation Activity and Selectivity of BM3 Variants usingSubstrates and Reaction Conditions Shown^(a)

entry enzyme TTN er 1 P411_(BM3)-CIS T438S 300 74:26 2 P450_(BM3)-CIST438S 7 nd 3 P411_(BM3)-CIS A268T T438S 19 nd 4 P411_(BM3)-H2-5-F10 14029:71 5 P411_(BM3)-H2-A-10 84 57:43 6 P411_(BM3)-H2-4-D4 32 70:30 7P450_(BM3) 10 nd 8 P411_(BM3) 11 nd 9 P450_(BM3)-T268A 19 nd 10P411_(BM3)-T268A 17 nd 11 P411_(BM3)-CIS I263A T438S 320 18:82^(a)“P411” denotes Ser-ligated (C400S) variant of cytochrome P450BM3.Variant IDs and specific amino acid substitutions in each can be foundin Table A. TTN—total turnover number, er = enantiomeric ratio, nd = notdetermined.

Since activating mutations T268A and C400S were already present inP411_(BM3)-CIS T438S, the effects of reverting each mutation to thewild-type residue (Table B, entries 1-3) were tested. Each revertant wasmuch less active than the parent, supporting the benefit of having theC400S and T268A mutations for effective nitrene-transfer chemistry.Given the bulky nature of the aryl sulfonylazide nitrene sources andaryl thioethers, the C400S mutants of several P450BM3 variants that hadbeen engineered via combinatorial alanine scanning to hydroxylate largesubstrates were tested (Table B, entries 4-6). While P411_(BM3)-H2-5-F10displayed comparably high levels of activity to P411_(BM3)-CIS T438S(>100 TTN), the other mutants we tested from this library were lessproductive. The effects of introducing the activating mutations intowild-type P450BM3 was also tested. Although these wild-type derivativeswere highly active and stereoselective for intramolecular C—H amination,neither single mutant (T268A or C400S) nor the double mutant(T268A+C400S) were as active for intermolecular sulfimidation.

The turnover data presented above demonstrate the sequence dependence ofsulfimidation productivity. The effects, however, could be due tochanges in stability of the enzymes that lead to degradation over thecourse of the reaction. To address this possibility, the initial ratesof reaction using the most productive enzyme in terms of total turnoverwere compared, P411BM3-CIS T438S, and the less productiveP411BM3-H2-A-10 and P411BM3-H2-4-D4 enzymes (FIG. 7). Differences in thetotal productivity (i.e., TTN) for each enzyme are mirrored in theinitial rates of reaction, suggesting that specific amino acids proximalto the heme influence binding and orientation of the substrates toeffect catalytic rate enhancement.

The key role of active site architecture in guiding reaction trajectoryis further supported by the effects of amino acid substitutions on thereaction stereochemistry. Experiments shows that enzymes capable ofproducing an excess of either sulfimide enantiomer: e.g., P411BM3-CIST438S gave an er of 74:26, while expanded active site variantP411BM3-H2-5-F10 exhibited the opposite selectivity, giving 29:71 (FIG.8). Among the H2 mutants (which differ from P411BM3-CIS T438S by 3-5amino acid substitutions, Table A), H2-5-F10 was alone in containing theI263A mutation, suggesting this mutation is relevant for enantiomericinversion observed in the P411_(BM3)-H2-5-F10 variant. When the I263Amutation was placed in the P411BM3-CIS background, an even morepronounced inversion in selectivity was observed (er=18:82 for theP411_(BM3)-CIS I263A T438S variant, compared to 74:26 for P411_(BM3)-CIST438S). This enzymatic system not only induces asymmetry in sulfimideproducts but also provides tunability in which selectivity can beswitched with just a few mutations.

Previous studies of P450-catalyzed sulfoxidation as well asrhodium-catalyzed C—H amination suggest that the electronic propertiesof sulfide or alkyl acceptor substrates significantly impact reactivity.Thus, to better understand the mechanism of this new enzyme reaction,experiments were performed to establish how thioether electronicproperties affected enzyme-catalyzed sulfimidation. A set of arylsulfide substrates with substituents encompassing a range of electronicproperties, from strongly donating to weakly withdrawing, were selected.As a first approximation of the effect of sulfide electronics, the totalnumber of turnovers catalyzed by P411_(BM3)-CIS T438S was determined inthe reactions of different sulfides with tosyl azide (Table C). Ingeneral, sulfides containing electron-donating substituents on the arylsulfide ring were better substrates for sulfimidation. For example, theenzyme reaction containing 4-methoxythioanisole methoxythioanisole (7a)gave the highest levels of activity (300 TTN). In contrast, theelectron-deficient p-aldehyde substrate (7e) gave only trace amounts ofsulfimide product. Further, some azides that initially appeared entirelyinactive gave small amounts of sulfimide products when reacted with4-methoxythioanisole, underscoring the importance of sulfide electronicsin this reaction (Table S4). The identity of substrates also exerted amodest influence on the enantioselectivity of sulfimidation. Inparticular, P411BM3-CIS T438S gave er values for substrates 8a-8d thatranged from 59:41 for 8c to 87:13 for 8d (Table S5, FIGS. S4-S6). Whileit is possible that some sulfides were poorer substrates due to thesteric influence of the para substituent, the overall trend is stronglysuggestive of electron induction to the aryl sulfide being a majorcontributor to activity. One notable aspect of these reactions is thatsignificantly more sulfonamide (9) was produced when less reactivesulfides were used.

TABLE C Impact of Sulfide Substituents on Sulfimidation Activity withP411_(BM3)-CIS T438S.

entry R₁ in 7 R₂ in 7 TTN 8 TTN 9 a —OMe —H 300 270 b —Me —H 190 400 c—H —Me 100 390 d —H —H  30 500 e —CHO —H  <1^(a) 510 ^(a)Trace productobserved by liquid chromatography-mass spectrometry (LC-MS).

Although the total turnover data suggest that sulfide electronicsinfluence reactivity, this result could also be due to other factors,such as substrate-dependent enzyme inactivation. To assess the effect ofsulfide substituents on reactivity more directly, the initial rates ofreaction of tosyl azide with the sulfides 7a-7d in Table 2 weremeasured. The initial rates correlated well with the total turnover datapresented above, with p-OMe showing the highest rate of reaction (FIG.12). Given this correlation, more mechanistic information was obtainedby fitting the data to a Hammett plot that correlates the observed rateswith each substituent's Hammett parameter. There was a strong, linearrelationship with a Hammett value of p=−4.0 (FIG. 2), which suggeststhat during the rate-limiting step there is a buildup of positive chargeon the sulfide that is stabilized by electron-donating substituents.This observation is consistent with Hammett values obtained for theoxidation of thioanisoles in P450-catalyzed sulfoxidation reactions,though the magnitude of p for sulfimidation is significantly greaterthan for sulfoxidation (−4.0 versus −0.2). One possible explanation forthis difference is that the presumed nitrenoid intermediate of thisreaction (FIG. 1) is a weaker oxidant than compound I, making thenucleophilicity of sulfur an important contributor to the rate ofnitrenoid transfer. The large difference in the magnitude of p couldalso indicates a change in mechanism relative to P450 sulfoxidation:whereas sulfimidation may occur via direct nucleophilic attack of thethioether on the nitrenoid intermediate.

As noted above, a greater proportion of sulfonamide side product wasformed when less reactive sulfides were used. The varying amounts ofthis side product prompted an examination of how sulfonamide might beproduced. Experiments were performed to test the possibility that azideis reduced by some additive in the reactions (i.e., glucose oxidase,catalase, NADPH, etc.) by simply omitting the P450 enzyme from thereactions (Table 7). No-enzyme controls yielded very little reducedsulfonamide product (more than 10-fold lower than with enzyme present).While these experiments showed that enzyme was likely involved in azidereduction, this still left several possibilities. Since P411_(BM3)'sheme domain is fused to a reductase, there was the possibility thatazide reduction occurs via direct hydride transfer from the reductase,as has been observed for aldehyde reductions. Thus, a carbonmonoxide-inhibited reactions was used to investigate this possibility,since CO binding to the heme iron should have no effect on the reductasedomain. In the presence of CO, there was a significant decrease in thesulfonamide produced, suggesting that azide reduction occurs at theheme. Furthermore, only trace sulfonamide was observed when reactionswere conducted in the presence of oxygen, further supporting theinvolvement of reduced heme in azide reduction. Since all the availableevidence suggests that azide reduction and sulfimide formation bothoccur at the heme, the most parsimonious explanation is that bothreactions stem from a common intermediate that can give rise to bothsulfonamide and sulfimide products.

A proposed mechanism of sulfonamide and sulfimide formation begins withthe iron(III) heme gaining an electron from NADPH via the flavincofactors of the reductase domain (FIG. 3). Addition of azide substrateresults in formation of a formal iron(IV) nitrenoid, which can either bereduced by subsequent electron transfer or “trapped” by sulfide to formsulfimide product. A second electron transfer followed by protonation ofthe nitrenoid is proposed to generate sulfonamide which restores theheme iron to its ferric state, and additional reductant is required toreturn to the catalytically active ferrous state (FIG. 3, unproductivepathway).

To test whether ferric heme is involved in the unproductive pathway, thechange in the visible absorbance spectrum of the reduced holoenzymeP411_(BM3)-CIS I263A T438S was monitored upon addition of NADPH followedby azide. The Ser-ligated P411 proteins exhibit different absorbanceproperties in the ferric and ferrous states compared with theirCys-ligated counterparts, such that the ferric, ferrous, and CO-ferrousSoret bands are shifted from 418, 408, and 450 nm to 405, 422, and 411nm, respectively (FIG. 12). When NADPH was added to a solution of enzymeunder an anaerobic atmosphere, a reduction of the heme from the ferricto the ferrous state was observed. When a degassed solution of azide wasadded to the ferrous protein, an immediate shift back to the ferricstate was observed, with concomitant production of sulfonamide, verifiedby high-performance liquid chromatography (HPLC). This observationsuggests that the unproductive azide reduction pathway occurs readily inthe absence of sulfide and that, when provided only with azide, thecatalyst rests in the ferric state.

To determine the resting state of the P411 catalyst in sulfimidation,the above experiment were repeated in the presence of both sulfide andazide. Addition of sulfide to a solution of enzyme and NADPH results inno change in the Q or Soret bands, with the iron heme remaining in theferrous state. However, addition of azide to this solution causes theiron heme to shift to the ferric state. After 10 min, peakscorresponding to the ferrous heme begin to grow until the ferrous hemebecomes the dominant species at 30 min (FIG. 14). Both sulfonamide andsulfimide products are formed throughout the course of the reaction. Theobservations are consistent with the competing reaction pathwaysoutlined in FIG. 3 and suggest that the catalyst rests as a mixture offerric and ferrous hemes during sulfimidation. When the concentration ofazide is high, as it is at the beginning of the reaction, theunproductive pathway is favored, and a ferric resting state dominates.As azide is consumed, both the ferric and ferrous resting states can beobserved.

P450 monooxygenases are known to undergo an “oxidase uncoupling” sidereaction in which compound I is reduced by two electrons to give water,which bears some similarity to the process of azide reduction observedhere. One difference, however, is that only a single electron transferis required to attain a reactive state in nitrene-transfer chemistry.This stands in contrast to P450 monooxygenase chemistry, where thegeneration of compound I from O₂ requires the transfer of two electrons.Thus, one explanation for the relatively high proportion of reducedazide in these reactions is that the electron-transfer machinery inP450_(BM3) is evolved to carry out two-electron reductions. In the caseof nitrene transfer chemistry, reducing the ferric heme to the +2 stateallows nitrenoid formation, but a second electron transfer wouldgenerate an unreactive iron(III) sulfonamide complex, as proposed forintramolecular C—H amination. Coupled with the fact that lower sulfideconcentrations and less-reactive sulfides lead to increased azidereduction, these observations are consistent with the mechanismdiscussed above in which sulfimide formation competes with azidereduction. Since electron transfers from the reductase domain are quiterapid, relatively reactive sulfides can successfully compete withreduction to form sulfimide.

The mechanistic picture described above suggests that achieving higherlevels of sulfide occupancy in the active site should favor sulfimideformation and inhibit azide reduction. This could be achieved withtighter binding of the sulfide acceptor substrate or by increasing theconcentration of sulfide relative to azide. Accordingly, experimentswere performed with excess sulfide or slow addition of azide todetermine if increased sulfimide formation could be obtained relative tosulfonamide. Increasing the sulfide concentration decreased reduction ofazide to sulfonamide and improved the ratio of sulfimide to sulfonamide,from 0.6 (with 0.5 equiv sulfide) to 1.8 (with 4 equiv sulfide) (FIG.15, Table D). Slow azide addition slightly increased the TTN forsulfimide and decreased sulfonamide formation in a 2 h reaction (FIG.16). That higher concentrations of sulfide substrate improve sulfimideproduction suggests that protein engineering can be used to improve thebinding of sulfide acceptor substrates and could also produce stronggains in the desired activity. Indeed, the specific activities of theenzyme catalysts reported here compare favorably with enantioselectiveiron-based catalysts, which routinely require catalyst loadings of 10mol %. Furthermore, engineering the holoenzyme or reductase domain tofavor one electron transfers might improve the proportion of desiredproduct relative to azide reduction, which could allow reaction withmore challenging organic acceptor substrates.

TABLE D Production of sulfimide 8a and sulfonamide 9 in the presence ofvarying levels of sulfide acceptor substrate. Sulfide Azide TTN 8a TTN 90.5 eq   1 eq 71 110 1 eq 1 eq 97 100 2 eq 1 eq 110 93 3 eq 1 eq 130 854 eq 1 eq 140 80

A C400S mutation (as described herein) for sulfimidation can berationalized that the less electron-donating axial serine ligand in P411enzymes likely makes the nitrenoid species a more potent oxidant. Theimpact of sulfide substituents on sulfimide formation is also reflectedin the generation of sulfonamide side product, suggesting the nitrenoidundergoes rapid reduction and can be productively insert into reactivesulfides. Characterization of the redox state of the heme iron in thepresence and absence of nitrene source and sulfide acceptor supports theproposal that nitrenoid “overreduction” competes with productivesulfimide formation and that the former is a two-electron processresulting in regeneration of ferric heme. Another interesting aspect ofthis enzyme reaction is the use of an aryl sulfonylazide nitrene source.The ability of the enzyme to accept larger aryl substrates may bebeneficial for development of enantioselective intermolecularnitrene-transfer catalysts. Intermolecular nitrene transfer in the formof sulfimidation can now be added to the impressive array of cytochromeP450 enzymes.

General.

Unless otherwise noted, all chemicals and reagents for chemicalreactions were obtained from commercial suppliers (Sigma-Aldrich, VWR,Alfa Aesar) and used without further purification. Silica gelchromatography purifications were carried out using AMD Silica Gel 60,230-400 mesh. 1H spectra were recorded on a Varian Inova 500 MHzinstrument in CDCl3, and are referenced to the residual solvent peak.Synthetic reactions were monitored using thin layer chromatography(Merck 60 gel plates) using an UV-lamp for visualization.

Chromatography.

Analytical high-performance liquid chromatography (HPLC) was carried outusing an Agilent 1200 series, and a Kromasil 100 C18 column (PeekeScientific, 4.6×50 mm, 5 μm). Semi-preparative HPLC was performed usingan Agilent XDB-C18 (9.4×250 mm, 5 μm). Analytical chiral HPLC wasconducted using a supercritical fluid chromatography (SFC) system withisopropanol and liquid CO₂ as the mobile phase. Chiralcel OB-H and OJcolumns were used to separate sulfimide enantiomers (4.6×150 mm, 5 μM).Sulfides were all commercially available and sulfimide standards wereprepared as reported. e.r. values determined by dividing the major peakarea by the sum of the peak areas determined by SFC chromatography.

Cloning and Site-Directed Mutagenesis.

pET22b(+) was used as a cloning and expression vector for all enzymesdescribed in this study. Site-directed mutagenesis on P411BM3-CIS T438Sto generate P411BM3-CIS I263A T438S was performed using a modifiedQuickChange™ mutagenesis protocol. The PCR products were gel purified,digested with DpnI, and directly transformed into E. coli strain BL21(DE3).

Determination of P450 Concentration.

Concentration of P450/P411 enzymes was accomplished by quantifying theamount of free hemin present in purified protein using thepyridine/hemochrome assay.

Protein Expression and Purification.

Enzymes used in purified protein experiments were expressed in BL21(DE3)E. coli cultures transformed with plasmid encoding P450 or P411variants. Expression and purification was performed as describedelsewhere, except that the shake rate was lowered to 130 RPM duringexpression. Following expression, cells were pelleted and frozen at −20°C. For purification, frozen cells were resuspended in buffer A (20 mMtris, 20 mM imidazole, 100 mM NaCl, pH 7.5, 4 mL/g of cell wet weight)and disrupted by sonication (2×1 min, output control 5, 50% duty cycle;Sonicator 3000, Misonix, Inc.). To pellet insoluble material, lysateswere centrifuged at 24,000×g for 0.5 h at 4° C. Proteins were expressedin a construct containing a 6×-His tag and were consequently purifiedusing a nickel NTA column (5 mL HisTrap HP, GE Healthcare, Piscataway,N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). P450 orP411 enzymes were then eluted on a linear gradient from 100% buffer A 0%buffer B (20 mM tris, 300 mM imidazole, 100 mM NaCl, pH 7.5) to 100%buffer B over 10 column volumes (P450/P411 enzymes elute at around 80 mMimidazole). Fractions containing P450 or P411 enzymes were pooled,concentrated, and subjected to three exchanges of phosphate buffer (0.1M KPi pH 8.0) to remove excess salt and imidazole. Concentrated proteinswere aliquoted, flash-frozen on powdered dry ice, and stored at −20° C.until later use.

Typical Procedure for Small-Scale Sulfimidation Bioconversions UnderAnaerobic Conditions Using Purified Enzymes.

Small-scale reactions (400 μL) were conducted anaerobically in 2 mLcrimp vials. A solution of aryl sulfide in DMSO or methanol (100 mM, 10μL) was added to the reaction vial via syringe, followed by arylsulfonylazide (100 mM, 10 μL, DMSO). Final concentrations of the reagents weretypically: 2.5 mM aryl sulfide, 2.5 mM arylsulfonyl azide, 10 mM NADPH,25 mM glucose, 5-20 μM P450. To the vials were then added acetonitrile(460 μL) and internal standard (1,3,5 trimethoxybenzene, 10 mM in 10%DMSO/90% acetonitrile, 1 mM final concentration). This mixture was thentransferred to a microcentrifuge tube, and centrifuged at 17,000×g for 5minutes. A portion (20 μL) of the supernatant was then analyzed by HPLC.Sulfimide formation was quantified by comparison of integrated peakareas of internal standard (1,3,5-trimethoxy benzene, 1 mM or1,3,5-trichlorobenzene, 1 mM) and sulfimide at 220 nm to a calibrationcurve made using synthetically produced sulfimide and internal standard.Coefficients determined from standard curves were multiplied by adilution factor in order to obtain sulfimide concentrations in thereaction mixture. Standard curves and response factors for products8a-8d are presented in FIGS. 17-20. For chiral HPLC, the quenchedreaction mixture was extracted twice with ethyl acetate (2×350 μL),dried under a light argon stream and resuspended in acetonitrile (100μL).

Controls to Confirm the Enzymatic Sulfimidation Activity of VariantP411_(BM3)CIS T438S.

Small-scale reactions (400 μL total volume) were set up and worked up asdescribed above. For the reaction containing hemin as catalyst, 10 μL ofa hemin solution (1 mM in 50% DMSOH2O) was added to a finalconcentration of 25 TTNs were determined as described above and arepresented in Table 7. CS denotes ‘complete system’ in which allcomponents of the reactions as described above are present. Variationsfrom the complete system are denoted with a “−X” where X is thecomponent removed.

TABLE 7 Control experiments using substrate 7c yielding products 8c(sulfimide) and 9 (sulfonamide). CS = complete system. Condition TTN 8cTTN 9 P411_(BM3)-CIS T438S (CS) 90 300 CS-NADPH 1 2 CS + Na₂S₂O₄ + CO 240 CS boiled enzyme 1 53 CS aerobic 2.3 16 Hemin + Na₂S₂O₄ 0 42CS-P411_(BM3)-CIS T438S 0 21

Synthesis of Substrates and Standards.

All sulfimides presented in Table 8 were obtained form commercialsources (Sigma Aldrich, Alfa Aesar). Sulfmide standards were synthesizedusing known techniques.

TABLE 8 Impact of sulfide substituents on Sulfimidation activity withP411_(BM3)-CIS T438S.

entry R₁ in 7 R₂ in 7 TTN 8 TTN 9 a —OMe —H 300 270 b —Me —H 190 400 c—H —Me 100 390 d —H —H  30 500 e —CHO —H  <1^(a) 510 ^(a)Trace productobserved by liquid chromatography-mass spectrometry (LC-MS).

₁H NMR (500 MHz; CDCl₃): δ=7.71 (d, J=8.0 Hz, 2H), 7.61 (d, J=8.9 Hz,2H), 7.15 (d, J=8.35 Hz, 2H), 6.96 (d, J=8.9 Hz, 2H), 3.83 (s, 3H), 2.81(s, 3H), 2.34 (s, 3H).

1H NMR (500 MHz; CDCl3): δ=7.72 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.2 Hz,2H), 7.28 (d, J=7.3 Hz, 2H), 7.16 (d, J=8.1 Hz, 2H), 2.82 (s, 3H), 2.39(s, 3H), 2.35 (s, 3H)

1H NMR (500 MHz; CDCl3): δ=7.73 (d, J=8.0 Hz, 2H), 7.48-7.33 (m, 4H),7.17 (d, J=8.1 Hz, 2H), 2.82 (s, 3H), 2.36 (s, 3H), 2.35 (s, 3H)

1H NMR (500 MHz; CDCl3): δ=7.72 (d, J=7.8 Hz, 2H), 7.55-7.46 (m, 5H),7.16 (d, J=8.1 Hz, 2H), 2.83 (s, 3H), 2.34 (s, 3H)

The 1H NMR listings above for products 8a-8d matched those ofcharacterized compounds.

Determination of Initial Rates.

Four 2-ml vials were charged with a stir bar, 10× oxygen depletionsystem (40 μL), and a solution of enzyme prior to crimp sealing with asilicon septum. Once sealed, the headspace was flushed with argon for atleast 10 minutes. Concurrently, a sealed 6-mL vial charged with glucose(250 mM, 400 μL), NADPH (20 mM, 400 μL), and KPi (pH=8.0, 0.1 M, 2.6 mL)was sparged for 10 minutes with argon. After degassing was complete, 340μL of the reaction solution was transferred to the 2-mL vial viasyringe. Sulfide (100 mM, 10 μL) was added to all four 2-mL vialsfollowed quickly (less than 20 seconds) by tosyl azide (100 mM, 10 μL).The reactions were quenched at 1-2 minute intervals over 5-10 minutes bydecapping and adding acetonitrile (460 μL). After 5 minutes of stirring,the vials were charged with internal standard and the reaction mixtureswere transferred to 1.8 mL tubes, which were vortexed and centrifuged(14,000×g, 5 min). The supernatant was transferred to a vial foranalysis by HPLC. Initial rates are plotted for individual enzymesreferenced in FIG. 7, and for various substituted aryl sulfides in FIG.12.

Visible Absorbance Spectroscopy and Observation of Resting States.

To a semi-micro anaerobic cuvette, 8 μL of P411_(BM3)CIS I263A T438S(400 μM) was added. To obtain a spectrum of the ferric protein, 0.5 mLof degassed phosphate buffer was added to the cuvette and the visiblespectrum was recorded from 650 to 400 nm. To obtain a spectrum of theferrous protein, the cuvette was sealed with a cap equipped with rubbersepta and the headspace of the cuvette was purged with a gentle streamof Ar for 3 min. A solution of NADPH (5 mM) was added to a 6 mL crimpvial and made anaerobic by sparging with Ar for 5 min. The NADPHsolution (0.5 mL) was then added to the anaerobic cuvette containingprotein. Visible spectra of the protein sample are recorded until astable ferrous state is reached. Representative spectra of the Fe(III)-and Fe(II)-protein are shown below (FIG. 13).

To determine the resting state of the protein in the unproductivecatalytic cycle, a degassed solution of tosyl azide (2 μL, 400 mM inDMSO) was added to a fully reduced sample of ferrous protein. Thevisible spectrum of the protein shifted to the ferric heme immediatelyand remained unchanged for 20 min. Addition of an aliquot of organicsolvent of similar volume did not cause the observed change in ironoxidation state. At the end of 20 minutes (FIG. 13), the cuvette wasuncapped and the reaction mixture was worked up following the generalprocedure for small scale reactions. HPLC of the resulting solutionconfirmed that a substantial amount of azide is reduced to thecorresponding sulfonamide.

To determine the resting state of the protein during the sulfimidationreaction, a degassed solution of sulfide 7a (2 μL, 400 mM in DMSO) wasadded to the cuvette containing P411BM3CIS I263A T438S in the presenceof NADPH. A visible absorbance spectrum of the mixture was recorded toensure that the oxidation state of iron heme is unchanged. Next, adegassed solution of tosyl azide (2 μL, 400 mM in DMSO) was added to thecuvette. Visible absorbance spectra of the solution were recorded at 5,7, 12, 18, 22, 25 and 30 min (FIG. 14). The appearance of ferrous hemeis observed over time. HPLC confirmed formation of both sulfimide andsulfonamide.

Excess Sulfide and Azide Slow Addition Experiments.

To assess the impact of sulfide concentration on overall productivity ofreaction, sulfide was added to reaction ranging from 0.5 eq to 4 eqrelative to azide. 1 eq of azide denotes 1 mM in the small scalereactions described above. Results are plotted in FIG. S10 below as aratio of the TTN for sulfimide vs. TTN for sulfonamide. Slow additionwas accomplished by adding 1 μL of a 100 mM (100 nmol) tosyl azidesolution (DMSO) at 15 minute intervals to a reaction set up as describedpreviously with 0.4% catalyst loading, containing 2.5 mM 7a. Azide wasadded over 150 minutes until equimolar final concentrations of sulfideand azide were achieved. Results of the slow addition are presented inFIG. 16.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the disclosure.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for catalyzing the intermolecularinsertion of nitrogen into organosulfur substrates to produce a producthaving a new S-N bond, the method comprising: providing a mixturecomprising a nitrene source, an organosulfur substrate and a heme enzymecomprising the amino acid sequence of SEQ ID NO: 1 or an engineered hemeenzyme comprising the amino acid sequence of SEQ ID NO: 1 with theexception of at least one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, or all of the following amino acidsubstitutions in the amino acid sequence of SEQ ID NO: 1_(L) V78A, F87C,P142S, T175I, A184V, S226R, H236Q, T438S, E252G, A290V, L353V, I366V,C400X, T438S and E442K, wherein “X” is any amino acid other than Cys;and reacting said mixture for a time sufficient to form a product havinga new S-N bond.
 2. The method of claim 1, wherein said nitrene source isan azide.
 3. The method of claim 2, wherein said azide has the generalformula R¹—N₃, wherein R¹ is: (i) a substituted or unsubstituted aryl, asubstituted or unsubstituted aryl, —OR², or —NR², wherein R² is asubstituted or unsubstituted aryl, a substituted or unsubstituted alkyl;(ii) —SO₂R³, wherein R³ is a substituted or unsubstituted aryl, asubstituted or unsubstituted alkyl, —OR², or —NR², wherein R² are anyalkyl or aryl; (iii) —COR⁴, wherein R⁴ is a substituted or unsubstitutedaryl, a substituted or unsubstituted alkyl, —OR², or —NR², wherein R²are any alkyl or aryl; or (iv) —P(O)(OR⁵)(OR⁶), wherein R⁵ and R⁶ areindependently H, a substituted or unsubstituted aryl, a substituted orunsubstituted alkyl.
 4. The method of claim 3, wherein said azide has astructure selected from the group consisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalky, or aryl.
 5. The method of claim 1, wherein said nitrene source isselected from the group consisting of:

wherein R¹ is any alkyl, aryl, —OR, NR², wherein R, R² and R³ are anyalkyl, or aryl.
 6. The method of claim 1, wherein said product having anew S-N bond is an aliphatic amine and said nitrene source is tosylazide.
 7. The method of claim 1, wherein said product having a new S-Nbond is generated through a nitrenoid intermediate.
 8. The method ofclaim 1, further comprising expressing said heme enzyme in a bacterial,archaeal or fungal host organism.
 9. The method of claim 1, wherein saidengineered heme enzyme comprises a T268A mutation, a T438S mutationand/or a C400X mutation in the amino acid sequence of SEQ ID NO: 1,wherein X is any amino acid other than Cys.
 10. The method of claim 1,wherein said product having a new S-N bond is a compound of Formula Ia:

wherein R¹ is a sulfoxide, a carbonyl or a phosphonate; wherein R² is Hor any alkyl or aryl; and wherein R³ is H, O or an optionallysubstituted aryl group.
 11. The method of claim 10, wherein R¹ is asulfoxide of formula SO₂R⁵, wherein R⁵ is any alkyl, any aryl, —OR⁶ orNR⁷, wherein R⁶ and Ware any alkyl or any aryl.
 12. The method of claim10, wherein W is a phosphonate of formula P(O)(OR⁸)(OR⁹), wherein R⁸ andR⁹ are independently any aryl or any alkyl.